EP2971185A1 - Methods and compositions for weed control - Google Patents

Methods and compositions for weed control

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Publication number
EP2971185A1
EP2971185A1 EP14780250.8A EP14780250A EP2971185A1 EP 2971185 A1 EP2971185 A1 EP 2971185A1 EP 14780250 A EP14780250 A EP 14780250A EP 2971185 A1 EP2971185 A1 EP 2971185A1
Authority
EP
European Patent Office
Prior art keywords
herbicides
polynucleotide
group
polynucleotides
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP14780250.8A
Other languages
German (de)
French (fr)
Other versions
EP2971185A4 (en
Inventor
Nengbing Tao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monsanto Technology LLC
Original Assignee
Monsanto Technology LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Monsanto Technology LLC filed Critical Monsanto Technology LLC
Priority to EP19192942.1A priority Critical patent/EP3604535A3/en
Publication of EP2971185A1 publication Critical patent/EP2971185A1/en
Publication of EP2971185A4 publication Critical patent/EP2971185A4/en
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the invention relates generally to the field of weed management. More specifically, the invention relates to control of Sorghum weed species and compositions containing polynucleotide molecules. The invention further provides methods and compositions useful for Johnsongrass control.
  • Weeds are plants that compete with cultivated plants in an agronomic environment and cost farmers billions of dollars annually in crop losses and the expense of efforts to keep weeds under control. Weeds also serve as hosts for crop diseases and insect pests. Weeds are plants that are unwanted in any particular environment. The losses caused by weeds in agricultural production environments include decreases in crop yield, reduced crop quality, increased irrigation costs, increased harvesting costs, reduced land value, injury to livestock, and crop damage from insects and diseases harbored by the weeds.
  • weeds cause these effects are: 1) competing with crop plants for water, nutrients, sunlight and other essentials for growth and development, 2) production of toxic or irritant chemicals that cause human or animal health problem, 3) production of immense quantities of seed or vegetative reproductive parts or both that contaminate agricultural products and perpetuate the species in agricultural lands, and 4) production on agricultural and
  • Herbicide tolerant weeds are a problem with nearly all herbicides in use, there is a need to effectively manage these weeds.
  • HRAC Herbicide Resistance Action Committee
  • NAHRAC North American Herbicide Resistance Action Committee
  • WSSA Weed Science Society of America
  • Sorghum weed species especially, Johnsongrass ⁇ Sorghum halepense) shattercane ⁇ Sorghum bicolor) and sudangrass ⁇ Sorghum Sudanese are difficult to control weeds that have been shown to develop tolerance to several classes of frequently used herbicides.
  • the present invention provides herbicidal compositions that comprise polynucleotide compositions useful for modulating gene expression in the Sorghum weed species, johnsongrass in particular, genes providing the production of herbicide target proteins, such as, acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS large subunit and ALS small subunit, also known as acetohydroxyacid synthase, AHAS), dihydropteroate synthetase (DHPS), 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), glutamine synthetase (GS2), 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), phytoene desaturase (PDS), protoporphyrinogen IX oxidase (PPOX) in plants for the purpose of enhancing control of johnsongrass in an agronomic environment and for the management of herbicide resistant johnsongrass.
  • the invention comprises a method of Sorghum species weed control, in particular johnsongrass ⁇ Sorghum halepense) plant control comprising an external application of a herbicidal composition to a Sorghum halepense plant or a part of the Sorghum halepense plant in need of control, said herbicidal composition comprising a polynucleotide, an organosilicone surfactant concentration of about 0.2 percent or greater, and an effective dose of a nonpolynucleotide herbicide, wherein the polynucleotide is at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120, wherein said treated plant is more sensitive to said nonpolynucleotide herbicide relative to a similar plant treated with a herbicide composition not containing said polynucleotide.
  • the herbicide composition comprises a
  • polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-25, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of aryloxyphenoxypropionates, cyclohexanediones and
  • the herbicide composition comprises a
  • polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 26-44, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
  • the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 45-59, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
  • the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 60-66, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonamides and asulam.
  • the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 67-74, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of glyphosate.
  • the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 75-89, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of glufosinate.
  • the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 90-96, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of triketones, isoxazoles, and pyrazoles.
  • the herbicide composition comprises a
  • polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 97-105, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of pyridazinones, pyridinecarboxamides, beflubutamid, fluridone, flurochloridone and flurtamone.
  • the herbicide composition comprises a
  • polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 106-120, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of acifluorfen-Na, bifenox, chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon, oxadiargyl, azafenidin, carfentra
  • the polynucleotide of the herbicide composition is at least 19 contiguous nucleotides, and at least 85 percent identical to a gene sequence selected from the group consisting of SEQ ID NO: 1-120.
  • the polynucleotide can also be sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
  • the herbicide composition comprises a
  • polynucleotide at least 19 contiguous nucleotide in length or at least 85 percent homologous to polynucleotides selected from the group consisting of SEQ ID NO: 121-386.
  • the polynucleotide can also be sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
  • the polynucleotide molecule containing composition of the invention may be combined with other herbicidal compounds in a premix or tankmix to provide additional control of unwanted johnsongrass plants in a field of crop plants or combined with other agricultural chemicals to provide additional benefit to crop plants in a field treated with the herbicide composition of the invention.
  • the invention provides a method and herbicide compositions containing a polynucleotide that provide for regulation of herbicide target gene expression and enhanced control of weedy Sorghum plant species and important herbicide resistant Sorghum weed biotypes. Aspects of the method can be applied to manage johnsongrass plants in agronomic and other cultivated environments.
  • Herbicide activity is often directed to known enzymes in a plant cell. These enzymes include acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS large subunit and ALS small subunit, also known as acetohydroxyacid synthase, AHAS), dihydropteroate synthetase (DHPS), 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), glutamine synthetase (GS2), 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), phytoene desaturase (PDS), and protoporphyrinogen IX oxidase (PPOX).
  • ACCase acetyl-CoA carboxylase
  • ALS large subunit and ALS small subunit also known as acetohydroxyacid synthase, AHAS
  • DHPS dihydropteroate synthetase
  • EPSPS 5
  • Plant genes encode for these enzymes and the polynucleotides that provide for the expression of these enzymes have been isolated from johnsongrass ⁇ Sorghum halepense) in the invention.
  • the genes that encode for these enzymes are herein referred to as herbicide target genes.
  • Acetyl-CoA carboxylase catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, this is the first and the committed step in the biosynthesis of long-chain fatty acids.
  • This enzyme is the target of many herbicides that include members of the chemical families of aryloxyphenoxypropionates,
  • cyclohexanediones and phenylpyrazoline that include, but are not limited to an
  • aryloxyphenoxypropionate comprising clodinafop (Propanoic acid, 2-[4-[(5-chloro-3-fluoro- 2-pyridinyl)oxy]phenoxy]-,2-propynyl ester, (2R)), cyhalofop (butyl(2R)-2-[4-(4-cyano-2- fluorophenoxy)phenoxy] propionate), diclofop (methyl 2-[4-(2,4- dichlorophenoxy)phenoxy]propanoate), fenoxaprop (ethyl (R)-2-[4-(6-chloro-l,3- benzoxazol-2-yloxy)phenoxy]propionate), fluazifop (2R)-2-[4-[[5-(trifluoromethyl)-2- pyridinyl]oxy]phenoxy]propanoic acid), haloxyfop (2-[4-[[[3-chloro-5-(tri
  • ALS acetolactate synthase, also known as acetohydroxyacid synthase, AHAS
  • AHAS acetohydroxyacid synthase
  • This enzyme is the target of many herbicides that include members of the chemical families of Sulfonylureas, Imidazolinones, Triazolopyrimidines,
  • Pyrimidinyl(thio)benzoates, and Sulfonylaminocarbonyl-triazolinones amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron- methyl-Na, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, metsulfuron- methyl , nicosulfuron, oxasulfuron , primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, tri
  • the dihydropteroate synthetase is an enzyme involved in folic acid synthesis which is needed for purine nucleotide biosynthesis. This enzyme is the target of herbicides that include the carbamate chemical family and sulfonamides and asulam.
  • the EPSPS (5 -enolpyruvylshikimate-3 -phosphate synthase) enzyme catalyzes the conversion of shikimate-3 -phosphate into 5 -enolpyruvyl-shikimate-3 -phosphate, an intermediate in the biochemical pathway for creating three essential aromatic amino acids (tyrosine, phenylalanine, and tryptophan).
  • the EPSPS enzyme is the target for the herbicide N-phosphonomethyl glycine also known as glyphosate.
  • the glutamine synthetase (GS2) enzyme is an essential enzyme in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine.
  • This enzyme is the target of phosphinic acids herbicides that include glufosinate-ammonium and bialaphos.
  • HPPD 4-hydroxyphenyl-pyruvate-dioxygenase
  • This enzyme is the target of many herbicides that include members of the chemical families of Triketones, Isoxazoles, and Pyrazoles, includes but are not limited to Triketones, such as, mesotrione, tefuryltrione, tembotrione, and sulcotrione; Isoxazoles, such as, isoxachlortole, pyrasulfotole, and isoxaflutole; Pyrazoles, such as, benzofenap, pyrazolynate, topramezone and pyrazoxyfen. Additional HPPD inhibitors include benzobicyclon and bicyclopyrone,
  • the phytoene desaturase (PDS) enzyme is an essential enzyme in the carotenoid biosysnthesis pathway. This enzyme is the target of herbicides that include Pyridazinones, Pyridinecarboxamides, beflubutamid, fluridone, flurochloridone and flurtamone.
  • PPOX Protoporphyrinogen oxidase catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules.
  • PPOX inhibitor herbicide which include but is not limited to acifluorfen-Na, bifenox, chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen- ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, pyra
  • solution refers to homogeneous mixtures and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions.
  • Weedy plants are plants that compete with cultivated plants, those of particular importance include, but are not limited to important invasive and noxious weeds and herbicide resistant biotypes in crop production, such as, Amaranthus species -A. albus, A. blitoides, A. hybridus, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. tuberculatus, and A. viridis; Ambrosia species - A. trifida, A. artemisifolia; Lolium species -L. multiflorum, L. rigidium, L perenne; Digitaria species -D. insularis; Euphorbia species -E.
  • important invasive and noxious weeds and herbicide resistant biotypes in crop production such as, Amaranthus species -A. albus, A. blitoides, A. hybridus, A. palmeri, A. powellii, A. retroflexus, A. spinos
  • Sorghum weed species include, but are not limited to johnsongrass (Sorghum halepense), shattercane (Sorghum biocolor), and sudangrass (Sorghum Sudanese).
  • the polynucleotide molecules of the invention were isolated from johnsongrass and may be applicable in the method and compositions to provide control of the sorghum weed species other than johnsongrass where sufficient homology and complementarity of the molecules exist.
  • the composition of the present invention will contain multiple polynucleotides and herbicides that include any one or more polynucleotides identical or complementary to a segment of the any one or more herbicide target gene sequences, and the corresponding nonpolynucleotide herbicides.
  • the composition may contain a pesticide, where the pesticide is selected from the group consisting of insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides. Any one or more of these compounds can be added to the trigger oligonucleotide to form a multi-component pesticide giving an even broader spectrum of agricultural protection.
  • insecticides such as abamectin, acephate, azinphos-methyl, bifenthrin, buprofezin, carbofuran, chlorfenapyr, chlorpyrifos, chlorpyrifos-methyl, cyfluthrin, beta- cyfluthrin, cyhalothrin, lambda-cyhalothrin, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, esfenvalerate, fenoxycarb, fenpropathrin, fenvalerate, fipronil, flucythrinate, tau-fluvalinate, fonophos, imidacloprid, isofenphos, malathion, metaldehyde, methamidophos, methidathion, methomyl, methopren
  • amino]carbonyl]indeno[ 1 ,2-e] [ 1 ,3,4]oxadiazine-4a(3H)-carboxylate (DPX-JW062), monocrotophos, oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, rotenone, sulprofos, tebufenozide, tefluthrin, terbufos, tetrachlorvinphos, thiodicarb, tralomethrin, trichlorfon and triflumuron; most preferably a glyphosate compound is formulated with a fungicide compound or combinations of fungicides, such as azoxystrobin, benomyl, blasticidin-S, Bordeaux mixture (tribasic copper sulfate), bromuconazole
  • fungicides are common for example, cyproconazole and azoxystrobin, difenoconazole, and metalaxyl-M, fludioxonil and metalaxyl-M, mancozeb and metalaxyl-M, copper hydroxide and metalaxyl-M, cyprodinil and fludioxonil, cyproconazole and propiconazole;
  • commercially available fungicide formulations for control of Asian soybean rust disease include, but are not limited to Quadris® (Syngenta Corp), Bravo® (Syngenta Corp), Echo 720® (Sipcam Agro Inc), Headline® 2.09EC (BASF Corp), Tilt® 3.6EC (Syngenta Corp), PropiMaxTM 3.6EC (Dow AgroSciences), Bumper® 41.8EC
  • glyphosate compositions as described in the present invention to provide enhanced protection from soybean rust disease; nematocides such as aldoxycarb and fenamiphos; bactericides such as streptomycin; acaricides such as amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; and biological agents such as Bacillus thuringiensis, Bacillus thuringiensis delta
  • Nonpolynucleotide herbicides are available that can be added to the composition of the present invention, for example, members of the herbicide families that include but are not limited to amide herbicides, aromatic acid herbicides, arsenical herbicides, benzothiazole herbicides, benzoylcyclohexanedione herbicides, benzofuranyl alkylsulfonate herbicides, carbamate herbicides, cyclohexene oxime herbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides, dinitroaniline herbicides, dinitrophenol herbicides, diphenyl ether herbicides, dithiocarbamate herbicides, halogenated aliphatic herbicides, imidazolinone herbicides, inorganic herbicides, nitrile herbicides, organophosphor o us herbicides, oxadiazolone herbicides, oxazole herbicides, ,
  • phenylenediamine herbicides pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides, pyridine herbicides, pyrimidinediamine herbicides, pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides, thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides, triazine herbicides, triazinone herbicides, triazole herbicides, triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides, and urea herbicides.
  • the rates of use of the added herbicides can be reduced in compositions comprising the polynucleotides of the invention.
  • Use rate reductions of the additional added herbicides can be 10-25 percent, 26-50 percent, 51-75 percent or more can be achieved that enhance the activity of the polynucleotides and herbicide composition and is contemplated as an aspect of the invention.
  • An agronomic field in need of johnsongrass plant control is treated by application of the herbicide composition of the present invention directly to the surface of the growing plants, such as by a spray.
  • the method is applied to control johnsongrass in a field of crop plants by spraying the field with the composition.
  • the composition can be provided as a tank mix, a sequential treatment of components (generally the polynucleotide containing composition followed by the herbicide), or a simultaneous treatment or mixing of one or more of the components of the composition from separate containers.
  • Treatment of the field can occur as often as needed to provide weed control and the components of the composition can be adjusted to target specific johnsongrass herbicide target genes through utilization of specific polynucleotides or polynucleotide compositions identical or complementary to the gene sequences.
  • the composition can be applied at effective use rates according to the time of application to the field, for example, preplant, at planting, post planting, post-harvest.
  • the nonpolynucleotide herbicides can be applied to a field at effective rates of 1 to 2000 g ai/ha (active ingredient per hectare) or more.
  • the polynucleotides of the composition can be applied at rates of 1 to 30 grams per acre depending on the number of polynucleotide molecules as needed for effective johnsongrass control.
  • Crop plants in which johnsongrass weed control is needed include but are not limited to, i) corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; or , iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or
  • an ornamental flowering plant or shrub or turf grass g., an ornamental flowering plant or shrub or turf grass.
  • the methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i. e., a plant not grown from a seed) include fruit trees and plants that include, but are not limited to, citrus, apples, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants.
  • the crop plants can be transgenic and genetically engineered or genetically selected to be resistant to one or more of the nonpolynucleotide herbicides.
  • DNA refers to a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule of genomic or synthetic origin, such as, a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule.
  • DNA sequence refers to the nucleotide sequence of a DNA molecule.
  • RNA refers to a single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) molecule of genomic or synthetic origin, such as, a polymer of ribonucleotide bases that comprise single or double stranded regions.
  • ssRNA single-stranded RNA
  • dsRNA double-stranded RNA
  • nucleotide sequences in the text of this specification are given, when read from left to right, in the 5 ' to 3 ' direction.
  • the nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations ⁇ 1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
  • polynucleotide refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of typically 50 or fewer nucleotides in length) and polynucleotides of 51 or more nucleotides.
  • Embodiments of this invention include compositions including oligonucleotides having a length of 19-25 nucleotides (19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24- mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 46, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or
  • a herbicide target gene comprises any polynucleotide molecule of the gene in a plant cell or fragment thereof for which the modulation of the expression of the herbicide target gene product is provided by the methods and compositions of the present invention.
  • a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.
  • Oligonucleotides and polynucleotides of the present invention can be made that are essentially identical or essentially complementary to adjacent genetic elements of a gene, for example, spanning the junction region of an intron and exon, the junction region of a promoter and a transcribed region, the junction region of a 5' leader and a coding sequence, the junction of a 3' untranslated region and a coding sequence.
  • Polynucleotide compositions used in the various embodiments of this invention include compositions including oligonucleotides or polynucleotides or a mixture of both, including R A or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof.
  • the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic
  • polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleo tides.
  • the synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleo tides.
  • polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or
  • the polynucleotide includes chemically modified nucleotides.
  • chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, US Patent Publication 20110171287, US Patent
  • modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (for example, fluorescein or rhodamine) or other label (for example, biotin).
  • a fluorescent moiety for example, fluorescein or rhodamine
  • other label for example, biotin
  • the polynucleotides can be single- or double-stranded RNA or single- or double- stranded DNA or double-stranded DNA/RNA hybrids or modified analogues thereof, and can be of oligonucleotide lengths or longer.
  • the polynucleotides that provide single-stranded RNA in the plant cell are selected from the group consisting of (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, and (f) a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (i) a
  • these polynucleotides include chemically modified nucleotides or non- canonical nucleotides.
  • the polynucleotides include double- stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization.
  • the polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having an at least partially double-stranded structure including at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression.
  • polynucleotides are or will produce single-stranded RNA with at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression.
  • the polynucleotides further includes a promoter, generally a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
  • gene refers to chromosomal DNA, plasmid DNA, cDNA, intron and exon DNA, artificial DNA polynucleotide, or other DNA that encodes a peptide, polypeptide, protein, or RNA transcript molecule, and the genetic elements flanking the coding sequence that are involved in the regulation of expression, such as, promoter regions, 5' leader regions, 3' untranslated regions. Any of the components of the herbicide target gene are potential targets for the oligonucleotides and polynucleotides of the present invention.
  • the polynucleotide molecules of the present invention are designed to modulate expression by inducing regulation or suppression of an endogenous herbicide target gene in a johnsongrass plant and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of the gene or to the sequence of RNA transcribed from the target gene, which can be coding sequence or non-coding sequence.
  • a trigger, or triggers effective polynucleotide molecules that modulate expression.
  • RNA transcribed RNA transcribed (known as messenger RNA or an RNA transcript) from the endogenous gene to effect regulation or suppression of expression of the endogenous gene.
  • Trigger molecules are identified by "tiling" the gene targets with partially overlapping probes or non-overlapping probes of antisense or sense polynucleotides that are essentially identical or essentially complementary to the nucleotide sequence of an endogenous gene. Multiple target sequences can be aligned and sequence regions with homology in common, according to the methods of the present invention, are identified as potential trigger molecules for the multiple targets.
  • trigger molecules of various lengths for example 19-25 nucleotides, 26-50 nucleotides, 51-100 nucleotides, 101-200 nucleotides, 201-300 nucleotides or more can be pooled into a few treatments in order to investigate polynucleotide molecules that cover a portion of a gene sequence (for example, a portion of a coding versus a portion of a noncoding region, or a 5 ' versus a 3 ' portion of a gene) or an entire gene sequence including coding and noncoding regions of a target gene.
  • Polynucleotide molecules of the pooled trigger molecules can be divided into smaller pools or single molecules inorder to identify trigger molecules that provide the desired effect.
  • the herbicide target gene R A and DNA polynucleotide molecules are sequenced by any number of available methods and equipment.
  • Some of the sequencing technologies are available commercially, such as the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif), as described below.
  • single molecule sequencing technologies are encompassed by the method of the invention and include the SMRT.TM. technology of Pacific Biosciences, the Ion Torrent.TM. technology, and nanopore sequencing being developed for example, by Oxford Nanopore Technologies.
  • Embodiments of single-stranded polynucleotides functional in this invention have sequence complementarity that need not be 100 percent, but is at least sufficient to permit hybridization to RNA transcribed from the herbicide target gene or DNA of the herbicide target gene to form a duplex to permit a gene silencing mechanism.
  • a polynucleotide fragment is designed to be essentially identical to, or essentially
  • RNA transcribed from the target gene By “essentially identical” is meant having 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene; by “essentially complementary” is meant having 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.
  • polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to one allele or one family member of a given target gene (coding or non-coding sequence of a gene for of the present invention); in other embodiments the polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
  • the polynucleotides used in the compositions that are essentially identical or essentially complementary to the target gene or transcript will comprise the predominant nucleic acid in the composition.
  • the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript will comprise at least about 50%, 75%, 95%, 98% or 100% of the nucleic acids provided in the composition by either mass or molar concentration.
  • the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to about 50%, about 10% to about 50%), about 20%> to about 50%>, or about 30%> to about 50%> of the nucleic acids provided in the composition by either mass or molar concentration.
  • compositions where the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1 % to 100%, about 10% to 100%, about 20% to about 100%, about 30% to about 50%, or about 50% to a 100% of the nucleic acids provided in the composition by either mass or molar concentration.
  • Identity refers to the degree of similarity between two polynucleic acid or protein sequences.
  • An alignment of the two sequences is performed by a suitable computer program.
  • a widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994).
  • the number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths.
  • the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
  • Trigger molecules for specific herbicide target gene family members can be identified from coding and/or non-coding sequences of gene families of a plant or multiple plants, by aligning and selecting 200-300 polynucleotide fragments from the least homologous regions amongst the aligned sequences and evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the herbicidal phenotype.
  • the effective segments are further subdivided into 50-60 polynucleotide fragments, prioritized by least homology, and reevaluated using topically applied polynucleotides.
  • the effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by least homology, and again evaluated for induction of the yield/quality phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or again evaluated in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
  • Trigger molecules for broad activity against Sorghum weed species can be identified from coding and/or non-coding sequences of gene families of a plant or multiple plants, by aligning and selecting 200-300 polynucleotide fragments from the most homologous regions amongst the aligned sequences and evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssR A, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the yield/quality phenotype.
  • the effective segments are subdivided into 50-60 polynucleotide fragments, prioritized by most homology, and reevaluated using topically applied polynucleotides.
  • the effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by most homology, and again evaluated for induction of the yield/quality phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
  • deoxyribonucleotides on the 3 ' end of the sense strand Long polynucleotide molecules can be synthesized from commercially available kits, for example, kits from Applied
  • Biosystems/Ambion have DNA ligated on the 5 ' end in a microbial expression cassette that includes a bacterial T7 polymerase promoter that makes RNA strands that can be assembled into a dsRNA and kits provided by various manufacturers that include T7
  • dsRNA molecules can be produced from microbial expression cassettes in bacterial cells (Ongvarrasopone et al. ScienceAsia 33:35-39; Yin, Appl. Microbiol. Biotechnol 84:323-333, 2009; Liu et al, BMC Biotechnology 10:85, 2010) that have regulated or deficient RNase III enzyme activity or the use of various viral vectors to produce sufficient quantities of dsRNA.
  • design parameters such as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326 - 330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676, 2006) are known in the art and are used in selecting polynucleotide sequences effective in gene silencing.
  • random design or empirical selection of polynucleotide sequences is used in selecting polynucleotide sequences effective in gene silencing.
  • the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
  • polynucleotide compositions of this invention are useful in compositions, such as solutions of polynucleotide molecules, at low concentrations, alone or in combination with other components either in the same solution or in separately applied solutions that provide a permeability-enhancing agent. While there is no upper limit on the concentrations and dosages of polynucleotide molecules that can useful in the methods of this invention, lower effective concentrations and dosages will generally be sought for efficiency.
  • a useful treatment for herbaceous plants using 25-mer oligonucleotide molecules is about 1 nanomole (nM) of oligonucleotide molecules per plant, for example, from about 0.05 to 1 nM per plant.
  • Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nM , or about 0.1 to about 20 nM, or about 1 nM to about 10 nM of polynucleotides per plant.
  • the factor IX when applied to oligonucleotide molecules is arbitrarily used to denote a treatment of 0.8 nM of polynucleotide molecule per plant; 10X, 8 nM of polynucleotide molecule per plant; and 100X, 80 nM of polynucleotide molecule per plant.
  • compositions of this invention are useful in compositions, such as liquids that comprise polynucleotide molecules, alone or in combination with other components either in the same liquid or in separately applied liquids that provide a transfer agent.
  • a transfer agent is an agent that, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enables the
  • a transfer agent is an agent that conditions the surface of plant tissue, e. g., leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotide molecules into plant cells.
  • the transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a
  • polynucleotide -transferring agent to the plant tissue.
  • the transferring agent is applied subsequent to the application of the polynucleotide composition.
  • the polynucleotide transfer agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and/or cell wall or membrane barriers into plant cells.
  • Suitable transfer agents to facilitate transfer of the polynucleotide into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides.
  • agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof.
  • Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof.
  • Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof.
  • polynucleotides include emulsions, reverse emulsions, liposomes, and other micellar-like compositions.
  • agents or treatments for conditioning of a plant to permeation by polynucleotides include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations.
  • Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in nonaqueous systems (such as is used in synthetic reactions).
  • Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e. g., plant-sourced oils, crop oils, such as those listed in the 9 th Compendium of Herbicide Adjuvants, can be used, e.
  • Transfer agents include, but are not limited to, organosilicone preparations.
  • an organosilicone preparation that is commercially available as Silwet® L-77 surfactant having CAS Number 27306-78-1 and EPA Number:
  • CAL.REG.NO. 5905-50073-AA can be used to prepare a polynucleotide composition.
  • a Silwet L-77 organosilicone preparation is used as a pre-spray treatment of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.
  • composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e.
  • any of the commercially available organosilicone is any of the commercially available organosilicone
  • a composition that comprises a polynucleotide molecule and an organosilicone preparation in the range of about 0.015 to about 2 percent by weight (wt percent) e. g. , about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) is used or provided.
  • wt percent percent by weight
  • Organosilicone preparations used in the methods and compositions provided herein can comprise one or more effective organosilicone compounds.
  • the phrase "effective organosilicone compound" is used to describe any organosilicone compound that is found in an organosilicone preparation that enables a polynucleotide to enter a plant cell.
  • an effective organosilicone compound can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of a target gene expression in the plant cell.
  • effective organosilicone compounds include, but are not limited to, compounds that can comprise: i) a trisiloxane head group that is covalently linked to, ii) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, iii) a poly glycol chain, that is covalently linked to, iv) a terminal group.
  • Trisiloxane head groups of such effective organosilicone compounds include, but are not limited to, heptamethyltrisiloxane.
  • Alkyl linkers can include, but are not limited to, an n- propyl linker.
  • Poly glycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Poly glycol chains can comprise a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14.
  • Terminal groups can include, but are not limited to, alkyl groups such as a methyl group.
  • Effective organosilicone compounds are believed to include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.
  • an organosilicone preparation that comprises an
  • organosilicone compound comprising a trisiloxane head group is used in the methods and compositions provided herein.
  • an organosilicone preparation that comprises an organosilicone compound comprising a heptamethyltrisiloxane head group is used in the methods and compositions provided herein.
  • an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein.
  • an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein.
  • compositions that comprises a polynucleotide molecule and one or more effective organosilicone compound in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) is used or provided.
  • Compositions of the present invention include but are not limited components that are one or more polynucleotides essentially identical to, or essentially complementary to herbicide target gene sequence (promota) of the range of about 0.015 to about
  • a transfer agent that provides for the polynucleotide to enter a plant cell
  • a herbicide that complements the action of the polynucleotide
  • one or more additional herbicides that further enhance the herbicide activity of the composition or provide an additional mode of action different from the complementing herbicide
  • various salts and stabilizing agents that enhance the utility of the composition as an admixture of the components of the composition.
  • methods include one or more applications of a
  • polynucleotide composition and one or more applications of a permeability-enhancing agent for conditioning of a plant to permeation by polynucleotides.
  • a permeability-enhancing agent for conditioning of a plant to permeation by polynucleotides.
  • embodiments of the polynucleotide molecules are double-stranded R A oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA polynucleotides, single- stranded RNA polynucleotides, double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides, double-stranded DNA polynucleotides, single-stranded DNA polynucleotides, chemically modified RNA or DNA oligonucleotides or polynucleotides or mixtures thereof.
  • a johnsongrass herbicide target gene includes coding
  • compositions of the invention can include polynucleotides and oligonucleotides designed to target multiple genes, or multiple segments of one or more genes.
  • the target gene can include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.
  • An aspect of the invention provides a method for modulating expression of an herbicide target gene in a johnsongrass plant including (a) conditioning of a plant to permeation by polynucleotides and (b) treatment of the plant with the polynucleotide molecules, wherein the polynucleotide molecules include at least one segment of 19 or more contiguous nucleotides cloned from or otherwise identified from the target gene in either anti- sense or sense orientation, whereby the polynucleotide molecules permeate the interior of the plant and induce modulation of the target gene.
  • the conditioning and polynucleotide application can be performed separately or in a single step.
  • the conditioning can precede or can follow the polynucleotide application within minutes, hours, or days. In some embodiments more than one conditioning step or more than one polynucleotide molecule application can be performed on the same plant.
  • the segment can be cloned or identified from (a) coding (protein-encoding), (b) non-coding (promoter and other gene related molecules), or (c) both coding and non-coding parts of the target gene.
  • Non-coding parts include DNA, such as promoter regions or the RNA transcribed by the DNA that provide RNA regulatory molecules, including but not limited to: introns, 5' or 3' untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, and riboswitches.
  • DNA such as promoter regions or the RNA transcribed by the DNA that provide RNA regulatory molecules, including but not limited to: introns, 5' or 3' untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs
  • Example 1 Polynucleotides related to the herbicide target genes of johnsongrass (Sorghum halepense)
  • ACCase acetyl-CoA carboxylase
  • ALS large subunit ALS small subunit
  • AHAS acetohydroxyacid synthase
  • DHPS dihydropteroate synthetase
  • EPSPS 5- enolpymvylshikimate-3 -phosphate synthase
  • GS2 glutamine synthetase
  • HPPD 4- hydroxyphenyl-pyruvate-dioxygenase
  • PDS phytoene desaturase
  • protoporphyrinogen IX oxidase PPOX
  • PPOX protoporphyrinogen IX oxidase
  • RNA molecules were extracted from johnsongrass tissues by methods standard in the field, for example, total RNA was extracted using Trizol Reagent (Invitrogen Corp, Carlsbad, CA Cat. No. 15596-018), following the manufacturer's protocol or modifications thereof by those skilled in the art of polynucleotide extraction that may enhance recover or purity of the extracted RNA. Briefly, starting with approximately 1 gram of ground plant tissue for extraction. Prealiquot 10 milliliters (mL) Trizol reagent to 15 mL conical tubes. Add ground powder to tubes and shake to homogenize. Incubate the homogenized samples for 5 minutes (min) at room temperature (RT) and then add 3 mL of chloroform.
  • Trizol Reagent Invitrogen Corp, Carlsbad, CA Cat. No. 15596-018
  • RNA pellet Dissolve the RNA pellet in approximately 200 microliters of Diethylpyrocarbonate (DEPC) treated water. Heat briefly at 65C to dissolve pellet and vortex or pipet to resuspend RNA pellet. Adjust RNA concentraiton to 1-2 microgram/microliter. RNA was used to make cDNA libraries by standard methods that were then sequenced.
  • DEPC Diethylpyrocarbonate
  • Genomic DNA was extracted using EZNA SP Plant DNA Mini kit (Omega Biotek, Norcross GA, Cat#D5511) and Lysing Matrix E tubes (Q-Biogen, Cat#6914), following the manufacturer's protocol or modifications thereof by those skilled in the art of polynucleotide extraction that may enhance recover or purity of the extracted DNA.
  • SOLiD (Applied Biosystems, ), and the Genome Analyzer (HiSeq2000, Illumina, San Diego, CA) are used to provide polynucleotide sequence from the DNA and RNA extracted from the plant tissues.
  • Raw sequence data is assembled into contigs.
  • the contig sequence is used to identify trigger molecules that can be applied to the plant to enable regulation of the gene expression.
  • SEQ ID NO: 1-120 (summarized in Table 1) contains the target cDNA and gDNA sequence contigs from the various herbicide target genes of johnsongrass.
  • Example 2 Polynucleotides of the invention related to trigger molecules of the johnsongrass herbicide target genes.
  • the gene sequences and fragments of SEQ ID NO: 1-120 were selected into short polynucleotide lengths of 30 contiguous nucleotides as shown in Table 2, SEQ ID NO: 121- 386. These polynucleotides are tested to select an efficacious trigger to any of the herbicide target gene sequence regions.
  • the trigger polynucleotides are constructed as sense or anti- sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids and combined with an organosilicone based transfer agent and nonpolynucleotide herbicide to provide a new herbicidal composition.
  • the polynucleotides are combined into sets of two to three polynucleotides per set, using 4-8 nM of each polynucleotide.
  • Each polynucleotide set is prepared with the organosilicone transfer agent and applied to a johnsongrass plant or to a field of crop plants containing johnsongrass plants in combination with a nonpolynucleotide herbicide that targets one or more of the enzymes of the herbicide target genes, or followed by the nonpolynucleotide herbicide treatment one to three days later.
  • the effect is measured as stunting the growth and/or killing of the plant and is measured 8-14 days after treatment with the herbicidal composition.
  • the most efficacious trigger sets are identified and the individual polynucleotides are tested in the same methods as the sets are and the most efficacious single polynucleotide is identified.
  • This method it is possible to identify one oligonucleotide or several oligonucleotides that effect plant sensitivity to nonpolynucleotide herbicide.
  • additional 19-30 polynucleotides can be selected from the sequences of SEQ ID NO: 1-120 that are specific for a herbicide target gene in johnsongrass or include activity against a few related weed species, for example, Sorghum bicolor and Sorghum Sudanese.
  • Example 3 Methods used in the invention related to treating plants or plant parts with a topical mixture of the trigger molecules.
  • Johnsongrass plants are grown in the greenhouse (30 / 20 C day/night T; 14 hour photoperiod) in 4 inch square pots containing Sun Gro® Redi-Earth and 3.5 kg/cubic meter Osmocote® 14-14-14 fertilizer.
  • ssDNA ro dsRNA targeting one or more of the herbicide target gene sequences from SEQ ID NO: 1-120 at 16 nM, formulated in 10 millimolar sodium phosphate buffer (pH 6.8) containing 2%
  • Plants are treated manually by pipetting 10 ⁇ ⁇ ⁇ polynucleotide solution on fully expanded mature leaves, for a total of 40 microliters of solution per plant. Twenty-four and forty-eight hours later, the plants are treated with and effective dose of the nonpolynucleotide herbicide corresponding to the herbicide target gene in which the polynucleotides have homology. Four replications of each treatment is conducted. Plant height is determined just before treatment and at intervals upto twelve days after herbicide treatments to determine effect of the polynucleotide and herbicide treatments.
  • Example 4 A method to control johnsongrass in a field.
  • polynucleotides that can modulate the expression of one or more herbicide target genes in johnsongrass In Table 2, an analysis of herbicide target gene sequences provided a collection of 30-mer polynucleotides that can be used in compositions to affect the growth or develop or sensitivity to a polynucleotide herbicide to control multiple weed species in a field. A composition containing 1 or 2 or 3 or 4 or more of the polynucleotides of Table 2 or fragments thereof would enable broad activity of the composition against the herbicide resistant johnsongrass species or multiple Sorghum weed species that occur in a field environment.
  • the method includes creating a composition that comprises components that include at least one polynucleotide of Table 2 or fragment thereof or any other effective gene expression modulating polynucleotide essentially identical or essentially complementary to SEQ ID NO: 1-120, a transfer agent that mobilizes the polynucleotide into a plant cell and nonpolynucleotide herbicide.
  • the polynucleotide of the composition includes a dsRNA, ssDNA or dsDNA or a combination thereof.
  • a composition containing a polynucleotide can have a use rate of about 1 to 30 grams or more per acre depending on the size of the polynucleotide and the number of polynucleotides in the composition.
  • the composition may include one or more additional herbicides as needed to provide effective multi-species weed control in addition to control of johnsongrass and related weed species.
  • a field of crop plants in need of weed plant control is treated by spray application of the composition.
  • the composition can be provided as a tank mix, a sequential treatment of components (generally the polynucleotide followed by the nonpolynucleotide herbicide), a simultaneous treatment or mixing of one or more of the components of the composition from separate containers or as a premix of all of the components of the herbicidal composition.
  • nonpolynucleotide herbicide families include but are not limited to amide herbicides, aromatic acid herbicides, arsenical herbicides, benzothiazole herbicides,
  • benzoylcyclohexanedione herbicides benzofuranyl alkylsulfonate herbicides, carbamate herbicides, cyclohexene oxime herbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides, dinitroaniline herbicides, dinitrophenol herbicides, diphenyl ether herbicides, dithiocarbamate herbicides, halogenated aliphatic herbicides, imidazolinone herbicides, inorganic herbicides, nitrile herbicides, organophosphor o us herbicides, oxadiazolone herbicides, oxazole herbicides, phenoxy herbicides, phenylenediamine herbicides, pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides, pyridine herbicides,
  • pyrimidinediamine herbicides pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides, thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides, triazine herbicides, triazinone herbicides, triazole herbicides, triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides, and urea herbicides.
  • Treatment of the plants in the field can occur as often as needed to provide weed control and the components of the composition can be adjusted to target specific weed species or weed families.

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Abstract

The present invention provides novel compositions for use to enhance weed control. Specifically, the present invention provides for methods and compositions that modulate gene expression in johnsongrass. The present invention also provides for combinations of compositions and methods that enhance johnsongrass control. The invention comprises a method of Sorghum species weed control, in particular johnsongrass (Sorghum halepense) plant control comprising an external application of a herbicidal composition to a Sorghum halepense plant or a part of the Sorghum halepense plant in need of control, said herbicidal composition comprising a polynucleotide, an organosilicone surfactant concentration of about 0.2 percent or greater, and an effective dose of a nonpolynucleotide herbicide.

Description

METHODS AND COMPOSITIONS FOR WEED CONTROL
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 61/779,476, filed March 13, 2013, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates generally to the field of weed management. More specifically, the invention relates to control of Sorghum weed species and compositions containing polynucleotide molecules. The invention further provides methods and compositions useful for Johnsongrass control.
BACKGROUND OF THE INVENTION
Weeds are plants that compete with cultivated plants in an agronomic environment and cost farmers billions of dollars annually in crop losses and the expense of efforts to keep weeds under control. Weeds also serve as hosts for crop diseases and insect pests. Weeds are plants that are unwanted in any particular environment. The losses caused by weeds in agricultural production environments include decreases in crop yield, reduced crop quality, increased irrigation costs, increased harvesting costs, reduced land value, injury to livestock, and crop damage from insects and diseases harbored by the weeds. The principal means by which weeds cause these effects are: 1) competing with crop plants for water, nutrients, sunlight and other essentials for growth and development, 2) production of toxic or irritant chemicals that cause human or animal health problem, 3) production of immense quantities of seed or vegetative reproductive parts or both that contaminate agricultural products and perpetuate the species in agricultural lands, and 4) production on agricultural and
nonagricultural lands of vast amounts of vegetation that must be disposed of. Herbicide tolerant weeds are a problem with nearly all herbicides in use, there is a need to effectively manage these weeds. There are over 365 weed biotypes currently identified as being herbicide resistant to one or more herbicides by the Herbicide Resistance Action Committee (HRAC), the North American Herbicide Resistance Action Committee (NAHRAC), and the Weed Science Society of America (WSSA).
Sorghum weed species, especially, Johnsongrass {Sorghum halepense) shattercane {Sorghum bicolor) and sudangrass {Sorghum Sudanese) are difficult to control weeds that have been shown to develop tolerance to several classes of frequently used herbicides.
The present invention provides herbicidal compositions that comprise polynucleotide compositions useful for modulating gene expression in the Sorghum weed species, johnsongrass in particular, genes providing the production of herbicide target proteins, such as, acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS large subunit and ALS small subunit, also known as acetohydroxyacid synthase, AHAS), dihydropteroate synthetase (DHPS), 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), glutamine synthetase (GS2), 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), phytoene desaturase (PDS), protoporphyrinogen IX oxidase (PPOX) in plants for the purpose of enhancing control of johnsongrass in an agronomic environment and for the management of herbicide resistant johnsongrass.
SUMMARY OF THE INVENTION
The invention comprises a method of Sorghum species weed control, in particular johnsongrass {Sorghum halepense) plant control comprising an external application of a herbicidal composition to a Sorghum halepense plant or a part of the Sorghum halepense plant in need of control, said herbicidal composition comprising a polynucleotide, an organosilicone surfactant concentration of about 0.2 percent or greater, and an effective dose of a nonpolynucleotide herbicide, wherein the polynucleotide is at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120, wherein said treated plant is more sensitive to said nonpolynucleotide herbicide relative to a similar plant treated with a herbicide composition not containing said polynucleotide.
In another aspect of the invention, the herbicide composition comprises a
polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-25, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of aryloxyphenoxypropionates, cyclohexanediones and
phenylpyrazoline.
In another aspect of the invention, the herbicide composition comprises a
polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 26-44, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
In another aspect of the invention, the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 45-59, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
In another aspect of the invention, the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 60-66, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of sulfonamides and asulam.
In another aspect of the invention, the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 67-74, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of glyphosate.
In another aspect of the invention, the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 75-89, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of glufosinate.
In another aspect of the invention, the herbicide composition comprises a polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 90-96, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of triketones, isoxazoles, and pyrazoles.
In another aspect of the invention, the herbicide composition comprises a
polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 97-105, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of pyridazinones, pyridinecarboxamides, beflubutamid, fluridone, flurochloridone and flurtamone.
In another aspect of the invention, the herbicide composition comprises a
polynucleotide at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 106-120, and an organosilicone surfactant concentration of about 0.2 percent or greater, and a nonpolynucleotide herbicide selected from the group consisting of acifluorfen-Na, bifenox, chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, pyrazogyl, and profluazol.
The polynucleotide of the herbicide composition is at least 19 contiguous nucleotides, and at least 85 percent identical to a gene sequence selected from the group consisting of SEQ ID NO: 1-120. The polynucleotide can also be sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
In another aspect of the invention, the herbicide composition comprises a
polynucleotide at least 19 contiguous nucleotide in length or at least 85 percent homologous to polynucleotides selected from the group consisting of SEQ ID NO: 121-386. The polynucleotide can also be sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
In a further aspect of the invention, the polynucleotide molecule containing composition of the invention may be combined with other herbicidal compounds in a premix or tankmix to provide additional control of unwanted johnsongrass plants in a field of crop plants or combined with other agricultural chemicals to provide additional benefit to crop plants in a field treated with the herbicide composition of the invention. DETAILED DESCRIPTION
The invention provides a method and herbicide compositions containing a polynucleotide that provide for regulation of herbicide target gene expression and enhanced control of weedy Sorghum plant species and important herbicide resistant Sorghum weed biotypes. Aspects of the method can be applied to manage johnsongrass plants in agronomic and other cultivated environments.
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.
Herbicide activity is often directed to known enzymes in a plant cell. These enzymes include acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS large subunit and ALS small subunit, also known as acetohydroxyacid synthase, AHAS), dihydropteroate synthetase (DHPS), 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), glutamine synthetase (GS2), 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), phytoene desaturase (PDS), and protoporphyrinogen IX oxidase (PPOX). Plant genes encode for these enzymes and the polynucleotides that provide for the expression of these enzymes have been isolated from johnsongrass {Sorghum halepense) in the invention. The genes that encode for these enzymes are herein referred to as herbicide target genes.
The Acetyl-CoA carboxylase (ACCase) enzyme catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, this is the first and the committed step in the biosynthesis of long-chain fatty acids. This enzyme is the target of many herbicides that include members of the chemical families of aryloxyphenoxypropionates,
cyclohexanediones and phenylpyrazoline, that include, but are not limited to an
aryloxyphenoxypropionate comprising clodinafop (Propanoic acid, 2-[4-[(5-chloro-3-fluoro- 2-pyridinyl)oxy]phenoxy]-,2-propynyl ester, (2R)), cyhalofop (butyl(2R)-2-[4-(4-cyano-2- fluorophenoxy)phenoxy] propionate), diclofop (methyl 2-[4-(2,4- dichlorophenoxy)phenoxy]propanoate), fenoxaprop (ethyl (R)-2-[4-(6-chloro-l,3- benzoxazol-2-yloxy)phenoxy]propionate), fluazifop (2R)-2-[4-[[5-(trifluoromethyl)-2- pyridinyl]oxy]phenoxy]propanoic acid), haloxyfop (2-[4-[[3-chloro-5-(trifluoromethyl)-2- pyridinyl]oxy]phenoxy]propanoic acid), propaquizafop (2-[[(l- methylethylidene)amino]oxy]ethyl (2R)-2-[4-[(6-chloro- 2quinoxalinyl)oxy]phenoxy]propanoate) and quizalofop(2R)-2-[4-[(6-chloro-2- quinoxalinyl)oxy]phenoxy]propanoic acid; a cyclohexanedione comprising alloxydim
(methyl 2,2-dimethyl-4,6-dioxo-5-[(lE)- 1 -[(2-propen- 1 - yloxy)imino]butyl]cyclohexanecarboxylate), butroxydim (2-[ 1 -(ethoxyimino)propyl]-3- hydroxy-5-[2,4,6-trimethyl-3-(l-oxobutyl)phenyl]-2-cyclohexen-l-one), clethodim (2-[l- [[[(2E)-3-chloro-2-propen-l-yl]oxy]imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2- cyclohexen- 1 -one), cycloxydim (2- [ 1 -(ethoxyimino)butyl] -3 -hydroxy-5 -(tetrahydro-2H- thiopyran-3-yl)-2-cyclohexen-l-one), profoxydim (2-[l-[[2-(4- chlorophenoxy)propoxy] imino]butyl] -3 -hydroxy-5 -(tetrahydro-2H-thiopyran-3 -yl)-2- cyclohexen- 1 -one), sethoxydim (2-[ 1 -(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3- hydroxy-2-cyclohexen- 1 -one), tepraloxydim (2-[ 1 -[[[(2E)-3-chloro-2-propen- 1 - yl]oxy]imino]propyl]-3-hydroxy-5-(tetrahydro-2H-pyran-4-yl)-2-cyclohexen- 1 -one) and tralkoxydim (2-[ 1 -(ethoxyimino)propyl]-3-hydroxy-5-(2,4,6-trimethylphenyl)-2-cyclohexen- 1-one); a phenylpyrazoline comprising pinoxaden (8-(2,6-diethyl-4-methylphenyl)-l,2,4,5- tetrahydro-7-oxo-7H-pyrazolo[ 1 ,2-d] [ 1 ,4,5]oxadiazepin-9-yl 2,2-dimethylpropanoate).
The ALS (acetolactate synthase, also known as acetohydroxyacid synthase, AHAS) enzyme catalyzes the first step in the synthesis of the branched-chain amino acids (valine, leucine, and isoleucine). This enzyme is the target of many herbicides that include members of the chemical families of Sulfonylureas, Imidazolinones, Triazolopyrimidines,
Pyrimidinyl(thio)benzoates, and Sulfonylaminocarbonyl-triazolinones, amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron- methyl-Na, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, metsulfuron- methyl , nicosulfuron, oxasulfuron , primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron, imazapic, imazamethabenz -methyl, imazamox, imazapyr, imazaquin, imazethapyr, cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, bispyribac-Na, pyribenzoxim, pyriftalid, pyrithiobac-Na, pyriminobac-methyl, flucarbazone-Na, and procarbazone-Na.
The dihydropteroate synthetase (DHPS) is an enzyme involved in folic acid synthesis which is needed for purine nucleotide biosynthesis. This enzyme is the target of herbicides that include the carbamate chemical family and sulfonamides and asulam.
The EPSPS (5 -enolpyruvylshikimate-3 -phosphate synthase) enzyme catalyzes the conversion of shikimate-3 -phosphate into 5 -enolpyruvyl-shikimate-3 -phosphate, an intermediate in the biochemical pathway for creating three essential aromatic amino acids (tyrosine, phenylalanine, and tryptophan). The EPSPS enzyme is the target for the herbicide N-phosphonomethyl glycine also known as glyphosate.
The glutamine synthetase (GS2) enzyme is an essential enzyme in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. This enzyme is the target of phosphinic acids herbicides that include glufosinate-ammonium and bialaphos.
The 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD) is an Fe-containing enzyme, that catalyzes the second reaction in the catabolism of tyrosine, the conversion of 4- hydroxyphenylpyruvate to homogentisate. This enzyme is the target of many herbicides that include members of the chemical families of Triketones, Isoxazoles, and Pyrazoles, includes but are not limited to Triketones, such as, mesotrione, tefuryltrione, tembotrione, and sulcotrione; Isoxazoles, such as, isoxachlortole, pyrasulfotole, and isoxaflutole; Pyrazoles, such as, benzofenap, pyrazolynate, topramezone and pyrazoxyfen. Additional HPPD inhibitors include benzobicyclon and bicyclopyrone,
The phytoene desaturase (PDS) enzyme is an essential enzyme in the carotenoid biosysnthesis pathway. This enzyme is the target of herbicides that include Pyridazinones, Pyridinecarboxamides, beflubutamid, fluridone, flurochloridone and flurtamone.
Protoporphyrinogen oxidase (PPOX) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. PPOX inhibitor herbicide, which include but is not limited to acifluorfen-Na, bifenox, chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen- ethyl, cinidon-ethyl, flumioxazin, flumiclorac-pentyl, fluthiacet-methyl, thidiazimin, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, pyrazogyl, and profluazol.
As used herein "solution" refers to homogeneous mixtures and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions.
Weedy plants are plants that compete with cultivated plants, those of particular importance include, but are not limited to important invasive and noxious weeds and herbicide resistant biotypes in crop production, such as, Amaranthus species -A. albus, A. blitoides, A. hybridus, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. tuberculatus, and A. viridis; Ambrosia species - A. trifida, A. artemisifolia; Lolium species -L. multiflorum, L. rigidium, L perenne; Digitaria species -D. insularis; Euphorbia species -E. heterophylla; Kochia species - K. scoparia; Sorghum species -S. halepense; Conyza species -C. bonariensis, C. canadensis, C. sumatrensis; Chloris species -C. truncate; Echinochola species - E. colona, E. crus-galli; Eleusine species -E. indica; Poa species -P. annua;
Plantago species -P. lanceolata; Avena species - A.fatua; Chenopodium species - C. album; Setaria species - S. viridis, Abutilon theophrasti, Ipomoea species, Sesbania, species, Cassia species, Sida species, Brachiaria, species and Solanum species.
Sorghum weed species include, but are not limited to johnsongrass (Sorghum halepense), shattercane (Sorghum biocolor), and sudangrass (Sorghum Sudanese). The polynucleotide molecules of the invention were isolated from johnsongrass and may be applicable in the method and compositions to provide control of the sorghum weed species other than johnsongrass where sufficient homology and complementarity of the molecules exist.
It is contemplated that the composition of the present invention will contain multiple polynucleotides and herbicides that include any one or more polynucleotides identical or complementary to a segment of the any one or more herbicide target gene sequences, and the corresponding nonpolynucleotide herbicides. Additionally, the composition may contain a pesticide, where the pesticide is selected from the group consisting of insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides. Any one or more of these compounds can be added to the trigger oligonucleotide to form a multi-component pesticide giving an even broader spectrum of agricultural protection.
Examples of such agricultural protectants with which compounds of this invention can be formulated are: insecticides such as abamectin, acephate, azinphos-methyl, bifenthrin, buprofezin, carbofuran, chlorfenapyr, chlorpyrifos, chlorpyrifos-methyl, cyfluthrin, beta- cyfluthrin, cyhalothrin, lambda-cyhalothrin, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, esfenvalerate, fenoxycarb, fenpropathrin, fenvalerate, fipronil, flucythrinate, tau-fluvalinate, fonophos, imidacloprid, isofenphos, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, methyl 7-chloro-2,5- dihydro-2-[[N-(methoxycarbonyl)-N-[4-(trifluoromethoxy)phenyl
]amino]carbonyl]indeno[ 1 ,2-e] [ 1 ,3,4]oxadiazine-4a(3H)-carboxylate (DPX-JW062), monocrotophos, oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, rotenone, sulprofos, tebufenozide, tefluthrin, terbufos, tetrachlorvinphos, thiodicarb, tralomethrin, trichlorfon and triflumuron; most preferably a glyphosate compound is formulated with a fungicide compound or combinations of fungicides, such as azoxystrobin, benomyl, blasticidin-S, Bordeaux mixture (tribasic copper sulfate), bromuconazole, captafol, captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride, copper salts, cymoxanil, cyproconazole, cyprodinil (CGA 219417), diclomezine, dicloran, difenoconazole, dimethomorph, diniconazole, diniconazole- M, dodine, edifenphos, epoxiconazole (BAS 480F), famoxadone, fenarimol, fenbuconazole, fenpiclonil, fenpropidin, fenpropimorph, fluazinam, fluquinconazole, flusilazole, flutolanil, flutriafol, folpet, fosetyl-aluminum, furalaxyl, hexaconazole, ipconazole, iprobenfos, iprodione, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb, mepronil, metalaxyl, metconazole, S-methyl 7-benzothiazolecarbothioate (CGA 245704), myclobutanil, neo-asozin (ferric methanearsonate), oxadixyl, penconazole, pencycuron, probenazole, prochloraz, propiconazole, pyrifenox, pyroquilon, quinoxyfen, spiroxamine (KWG4168), sulfur, tebuconazole, tetraconazole, thiabendazole, thiophanate-methyl, thiram, triadimefon, triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycin and vinclozolin;
combinations of fungicides are common for example, cyproconazole and azoxystrobin, difenoconazole, and metalaxyl-M, fludioxonil and metalaxyl-M, mancozeb and metalaxyl-M, copper hydroxide and metalaxyl-M, cyprodinil and fludioxonil, cyproconazole and propiconazole; commercially available fungicide formulations for control of Asian soybean rust disease include, but are not limited to Quadris® (Syngenta Corp), Bravo® (Syngenta Corp), Echo 720® (Sipcam Agro Inc), Headline® 2.09EC (BASF Corp), Tilt® 3.6EC (Syngenta Corp), PropiMax™ 3.6EC (Dow AgroSciences), Bumper® 41.8EC
(MakhteshimAgan), Folicur® 3.6F (Bayer CropScience), Laredo® 25EC (Dow
AgroSciences), Laredo™ 25EW (Dow AgroSciences), Stratego® 2.08F (Bayer Corp), Domark™ 125SL (Sipcam Agro USA), and Pristine®38%WDG (BASF Corp) these can be combined with glyphosate compositions as described in the present invention to provide enhanced protection from soybean rust disease; nematocides such as aldoxycarb and fenamiphos; bactericides such as streptomycin; acaricides such as amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; and biological agents such as Bacillus thuringiensis, Bacillus thuringiensis delta endotoxin, baculovirus, and entomopathogenic bacteria, virus and fungi.
Numerous nonpolynucleotide herbicides are available that can be added to the composition of the present invention, for example, members of the herbicide families that include but are not limited to amide herbicides, aromatic acid herbicides, arsenical herbicides, benzothiazole herbicides, benzoylcyclohexanedione herbicides, benzofuranyl alkylsulfonate herbicides, carbamate herbicides, cyclohexene oxime herbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides, dinitroaniline herbicides, dinitrophenol herbicides, diphenyl ether herbicides, dithiocarbamate herbicides, halogenated aliphatic herbicides, imidazolinone herbicides, inorganic herbicides, nitrile herbicides, organophosphorous herbicides, oxadiazolone herbicides, oxazole herbicides, phenoxy herbicides,
phenylenediamine herbicides, pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides, pyridine herbicides, pyrimidinediamine herbicides, pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides, thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides, triazine herbicides, triazinone herbicides, triazole herbicides, triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides, and urea herbicides. In particular, the rates of use of the added herbicides can be reduced in compositions comprising the polynucleotides of the invention. Use rate reductions of the additional added herbicides can be 10-25 percent, 26-50 percent, 51-75 percent or more can be achieved that enhance the activity of the polynucleotides and herbicide composition and is contemplated as an aspect of the invention.
An agronomic field in need of johnsongrass plant control is treated by application of the herbicide composition of the present invention directly to the surface of the growing plants, such as by a spray. For example, the method is applied to control johnsongrass in a field of crop plants by spraying the field with the composition. The composition can be provided as a tank mix, a sequential treatment of components (generally the polynucleotide containing composition followed by the herbicide), or a simultaneous treatment or mixing of one or more of the components of the composition from separate containers. Treatment of the field can occur as often as needed to provide weed control and the components of the composition can be adjusted to target specific johnsongrass herbicide target genes through utilization of specific polynucleotides or polynucleotide compositions identical or complementary to the gene sequences. The composition can be applied at effective use rates according to the time of application to the field, for example, preplant, at planting, post planting, post-harvest. The nonpolynucleotide herbicides can be applied to a field at effective rates of 1 to 2000 g ai/ha (active ingredient per hectare) or more. The polynucleotides of the composition can be applied at rates of 1 to 30 grams per acre depending on the number of polynucleotide molecules as needed for effective johnsongrass control.
Crop plants in which johnsongrass weed control is needed include but are not limited to, i) corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; or , iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; or, vi) an ornamental plant (e. g., an ornamental flowering plant or shrub or turf grass). The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i. e., a plant not grown from a seed) include fruit trees and plants that include, but are not limited to, citrus, apples, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants. The crop plants can be transgenic and genetically engineered or genetically selected to be resistant to one or more of the nonpolynucleotide herbicides.
Polynucleotides
As used herein, the term "DNA", "DNA molecule", "DNA polynucleotide molecule" refers to a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule of genomic or synthetic origin, such as, a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule. As used herein, the term "DNA sequence", "DNA nucleotide sequence" or "DNA polynucleotide sequence" refers to the nucleotide sequence of a DNA molecule. As used herein, the term "RNA", "RNA molecule", "RNA polynucleotide molecule" refers to a single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) molecule of genomic or synthetic origin, such as, a polymer of ribonucleotide bases that comprise single or double stranded regions. Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5 ' to 3 ' direction. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations § 1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, "polynucleotide" refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to "oligonucleotides" (a polynucleotide molecule of typically 50 or fewer nucleotides in length) and polynucleotides of 51 or more nucleotides. Embodiments of this invention include compositions including oligonucleotides having a length of 19-25 nucleotides (19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24- mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 46, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (for example, polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a herbicide target gene including coding or non-coding or both coding and non-coding portions of the target gene). A herbicide target gene comprises any polynucleotide molecule of the gene in a plant cell or fragment thereof for which the modulation of the expression of the herbicide target gene product is provided by the methods and compositions of the present invention. Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs. Oligonucleotides and polynucleotides of the present invention can be made that are essentially identical or essentially complementary to adjacent genetic elements of a gene, for example, spanning the junction region of an intron and exon, the junction region of a promoter and a transcribed region, the junction region of a 5' leader and a coding sequence, the junction of a 3' untranslated region and a coding sequence.
Polynucleotide compositions used in the various embodiments of this invention include compositions including oligonucleotides or polynucleotides or a mixture of both, including R A or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof. In some embodiments, the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic
polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleo tides. In some embodiments, the
polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or
pseudouridine. In some embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, US Patent Publication 20110171287, US Patent
Publication 20110171176, and US Patent Publication 20110152353, US Patent Publication, 20110152346, US Patent Publication 20110160082, herein incorporated by reference. For example, including but not limited to the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide can be partially or completely modified with
phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (for example, fluorescein or rhodamine) or other label (for example, biotin).
The polynucleotides can be single- or double-stranded RNA or single- or double- stranded DNA or double-stranded DNA/RNA hybrids or modified analogues thereof, and can be of oligonucleotide lengths or longer. In more specific embodiments of the invention the polynucleotides that provide single-stranded RNA in the plant cell are selected from the group consisting of (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, and (f) a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (i) a double-stranded, hybridized RNA/DNA molecule, or combinations thereof. In some embodiments these polynucleotides include chemically modified nucleotides or non- canonical nucleotides. In embodiments of the method the polynucleotides include double- stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In one embodiment the polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having an at least partially double-stranded structure including at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. Not intending to be bound by any mechanism, it is believed that such polynucleotides are or will produce single-stranded RNA with at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. In certain other embodiments the polynucleotides further includes a promoter, generally a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
The term "gene" refers to chromosomal DNA, plasmid DNA, cDNA, intron and exon DNA, artificial DNA polynucleotide, or other DNA that encodes a peptide, polypeptide, protein, or RNA transcript molecule, and the genetic elements flanking the coding sequence that are involved in the regulation of expression, such as, promoter regions, 5' leader regions, 3' untranslated regions. Any of the components of the herbicide target gene are potential targets for the oligonucleotides and polynucleotides of the present invention.
The polynucleotide molecules of the present invention are designed to modulate expression by inducing regulation or suppression of an endogenous herbicide target gene in a johnsongrass plant and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of the gene or to the sequence of RNA transcribed from the target gene, which can be coding sequence or non-coding sequence. These effective polynucleotide molecules that modulate expression are referred to as "a trigger, or triggers". By "essentially identical" or "essentially complementary" is meant that the trigger polynucleotides (or at least one strand of a double-stranded polynucleotide or portion thereof, or a portion of a single strand polynucleotide) are designed to hybridize to the endogenous gene noncoding sequence (including promoters and regulatory elements of the gene) or to RNA transcribed (known as messenger RNA or an RNA transcript) from the endogenous gene to effect regulation or suppression of expression of the endogenous gene. Trigger molecules are identified by "tiling" the gene targets with partially overlapping probes or non-overlapping probes of antisense or sense polynucleotides that are essentially identical or essentially complementary to the nucleotide sequence of an endogenous gene. Multiple target sequences can be aligned and sequence regions with homology in common, according to the methods of the present invention, are identified as potential trigger molecules for the multiple targets. Multiple trigger molecules of various lengths, for example 19-25 nucleotides, 26-50 nucleotides, 51-100 nucleotides, 101-200 nucleotides, 201-300 nucleotides or more can be pooled into a few treatments in order to investigate polynucleotide molecules that cover a portion of a gene sequence (for example, a portion of a coding versus a portion of a noncoding region, or a 5 ' versus a 3 ' portion of a gene) or an entire gene sequence including coding and noncoding regions of a target gene. Polynucleotide molecules of the pooled trigger molecules can be divided into smaller pools or single molecules inorder to identify trigger molecules that provide the desired effect. The herbicide target gene R A and DNA polynucleotide molecules are sequenced by any number of available methods and equipment. Some of the sequencing technologies are available commercially, such as the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) and Helicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, Calif), as described below. In addition to the single molecule sequencing performed using sequencing-by-synthesis of Helicos Biosciences, other single molecule sequencing technologies are encompassed by the method of the invention and include the SMRT.TM. technology of Pacific Biosciences, the Ion Torrent.TM. technology, and nanopore sequencing being developed for example, by Oxford Nanopore Technologies.
Embodiments of single-stranded polynucleotides functional in this invention have sequence complementarity that need not be 100 percent, but is at least sufficient to permit hybridization to RNA transcribed from the herbicide target gene or DNA of the herbicide target gene to form a duplex to permit a gene silencing mechanism. Thus, in embodiments, a polynucleotide fragment is designed to be essentially identical to, or essentially
complementary to, a sequence of 19 or more contiguous nucleotides in either DNA gene sequence or messenger RNA transcribed from the target gene. By "essentially identical" is meant having 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene; by "essentially complementary" is meant having 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 19 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene. In some embodiments of this invention polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to one allele or one family member of a given target gene (coding or non-coding sequence of a gene for of the present invention); in other embodiments the polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
In certain embodiments, the polynucleotides used in the compositions that are essentially identical or essentially complementary to the target gene or transcript will comprise the predominant nucleic acid in the composition. Thus in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript will comprise at least about 50%, 75%, 95%, 98% or 100% of the nucleic acids provided in the composition by either mass or molar concentration. However, in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to about 50%, about 10% to about 50%), about 20%> to about 50%>, or about 30%> to about 50%> of the nucleic acids provided in the composition by either mass or molar concentration. Also provided are compositions where the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1 % to 100%, about 10% to 100%, about 20% to about 100%, about 30% to about 50%, or about 50% to a 100% of the nucleic acids provided in the composition by either mass or molar concentration.
"Identity" refers to the degree of similarity between two polynucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there are 100 matched amino acids between a 200 and a 400 amino acid protein, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
Trigger molecules for specific herbicide target gene family members can be identified from coding and/or non-coding sequences of gene families of a plant or multiple plants, by aligning and selecting 200-300 polynucleotide fragments from the least homologous regions amongst the aligned sequences and evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the herbicidal phenotype. The effective segments are further subdivided into 50-60 polynucleotide fragments, prioritized by least homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by least homology, and again evaluated for induction of the yield/quality phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or again evaluated in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
Trigger molecules for broad activity against Sorghum weed species can be identified from coding and/or non-coding sequences of gene families of a plant or multiple plants, by aligning and selecting 200-300 polynucleotide fragments from the most homologous regions amongst the aligned sequences and evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssR A, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the yield/quality phenotype. The effective segments are subdivided into 50-60 polynucleotide fragments, prioritized by most homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by most homology, and again evaluated for induction of the yield/quality phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
Methods of making polynucleotides are well known in the art. Chemical synthesis, in vivo synthesis and in vitro synthesis methods and compositions are known in the art and include various viral elements, microbial cells, modified polymerases, and modified nucleotides. Commercial preparation of oligonucleotides often provides two
deoxyribonucleotides on the 3 ' end of the sense strand. Long polynucleotide molecules can be synthesized from commercially available kits, for example, kits from Applied
Biosystems/Ambion (Austin, TX) have DNA ligated on the 5 ' end in a microbial expression cassette that includes a bacterial T7 polymerase promoter that makes RNA strands that can be assembled into a dsRNA and kits provided by various manufacturers that include T7
RiboMax Express (Promega, Madison, WI), AmpliScribe T7-Flash (Epicentre, Madison, WI), and TranscriptAid T7 High Yield (Fermentas, Glen Burnie, MD). dsRNA molecules can be produced from microbial expression cassettes in bacterial cells (Ongvarrasopone et al. ScienceAsia 33:35-39; Yin, Appl. Microbiol. Biotechnol 84:323-333, 2009; Liu et al, BMC Biotechnology 10:85, 2010) that have regulated or deficient RNase III enzyme activity or the use of various viral vectors to produce sufficient quantities of dsRNA. In some embodiments design parameters such as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326 - 330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676, 2006) are known in the art and are used in selecting polynucleotide sequences effective in gene silencing. In some embodiments random design or empirical selection of polynucleotide sequences is used in selecting polynucleotide sequences effective in gene silencing. In some embodiments the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
The polynucleotide compositions of this invention are useful in compositions, such as solutions of polynucleotide molecules, at low concentrations, alone or in combination with other components either in the same solution or in separately applied solutions that provide a permeability-enhancing agent. While there is no upper limit on the concentrations and dosages of polynucleotide molecules that can useful in the methods of this invention, lower effective concentrations and dosages will generally be sought for efficiency. The
concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, or seed. In one embodiment, a useful treatment for herbaceous plants using 25-mer oligonucleotide molecules is about 1 nanomole (nM) of oligonucleotide molecules per plant, for example, from about 0.05 to 1 nM per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nM , or about 0.1 to about 20 nM, or about 1 nM to about 10 nM of polynucleotides per plant. To illustrate embodiments of the invention, the factor IX, when applied to oligonucleotide molecules is arbitrarily used to denote a treatment of 0.8 nM of polynucleotide molecule per plant; 10X, 8 nM of polynucleotide molecule per plant; and 100X, 80 nM of polynucleotide molecule per plant.
The polynucleotide compositions of this invention are useful in compositions, such as liquids that comprise polynucleotide molecules, alone or in combination with other components either in the same liquid or in separately applied liquids that provide a transfer agent. As used herein, a transfer agent is an agent that, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enables the
polynucleotide to enter a plant cell. In certain embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e. g., leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotide molecules into plant cells. The transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a
polynucleotide -transferring agent to the plant tissue. In some embodiments the transferring agent is applied subsequent to the application of the polynucleotide composition. The polynucleotide transfer agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and/or cell wall or membrane barriers into plant cells. Suitable transfer agents to facilitate transfer of the polynucleotide into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof.
Embodiments of agents or treatments for conditioning of a plant to permeation by
polynucleotides include emulsions, reverse emulsions, liposomes, and other micellar-like compositions. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in nonaqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e. g., plant-sourced oils, crop oils, such as those listed in the 9th Compendium of Herbicide Adjuvants, can be used, e. g., paraffmic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. Transfer agents include, but are not limited to, organosilicone preparations.
In certain embodiments, an organosilicone preparation that is commercially available as Silwet® L-77 surfactant having CAS Number 27306-78-1 and EPA Number:
CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, New York can be used to prepare a polynucleotide composition. In certain embodiments where a Silwet L-77 organosilicone preparation is used as a pre-spray treatment of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) is used or provided.
In certain embodiments, any of the commercially available organosilicone
preparations provided such as the following Breakthru S 321 , Breakthru S 200 Cat# 67674- 67-3, Breakthru OE 441 Cat#68937-55-3, Breakthru S 278 Cat #27306-78-1 , Breakthru S 243, Breakthru S 233 Cat#134180-76-0, available from manufacturer Evonik Goldschmidt (Germany), Silwet® HS 429, Silwet® HS 312, Silwet® HS 508, Silwet® HS 604
(Momentive Performance Materials, Albany, New York) can be used as transfer agents in a polynucleotide composition. In certain embodiments where an organosilicone preparation is used as a pre-spray treatment of plant leaves or other surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) is used or provided.
Organosilicone preparations used in the methods and compositions provided herein can comprise one or more effective organosilicone compounds. As used herein, the phrase "effective organosilicone compound" is used to describe any organosilicone compound that is found in an organosilicone preparation that enables a polynucleotide to enter a plant cell. In certain embodiments, an effective organosilicone compound can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of a target gene expression in the plant cell. In general, effective organosilicone compounds include, but are not limited to, compounds that can comprise: i) a trisiloxane head group that is covalently linked to, ii) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, iii) a poly glycol chain, that is covalently linked to, iv) a terminal group.
Trisiloxane head groups of such effective organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n- propyl linker. Poly glycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Poly glycol chains can comprise a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Effective organosilicone compounds are believed to include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.
(Compound I: polyalkyleneoxide heptamethyltrisiloxane, average n=7.5). In certain embodiments, an organosilicone preparation that comprises an
organosilicone compound comprising a trisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a heptamethyltrisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and one or more effective organosilicone compound in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01 , 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.5 wt percent) is used or provided. Compositions of the present invention include but are not limited components that are one or more polynucleotides essentially identical to, or essentially complementary to herbicide target gene sequence (promoter, intron, exon, 5 ' untranslated region, 3 '
untranslated region), a transfer agent that provides for the polynucleotide to enter a plant cell, a herbicide that complements the action of the polynucleotide, one or more additional herbicides that further enhance the herbicide activity of the composition or provide an additional mode of action different from the complementing herbicide, various salts and stabilizing agents that enhance the utility of the composition as an admixture of the components of the composition.
In aspects of the invention, methods include one or more applications of a
polynucleotide composition and one or more applications of a permeability-enhancing agent for conditioning of a plant to permeation by polynucleotides. When the agent for
conditioning to permeation is an organosilicone composition or compound contained therein, embodiments of the polynucleotide molecules are double-stranded R A oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA polynucleotides, single- stranded RNA polynucleotides, double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides, double-stranded DNA polynucleotides, single-stranded DNA polynucleotides, chemically modified RNA or DNA oligonucleotides or polynucleotides or mixtures thereof.
In various embodiments, a johnsongrass herbicide target gene includes coding
(protein-coding or translatable) sequence, non-coding (non-translatable) sequence, or both coding and non-coding sequence. Compositions of the invention can include polynucleotides and oligonucleotides designed to target multiple genes, or multiple segments of one or more genes. The target gene can include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.
An aspect of the invention provides a method for modulating expression of an herbicide target gene in a johnsongrass plant including (a) conditioning of a plant to permeation by polynucleotides and (b) treatment of the plant with the polynucleotide molecules, wherein the polynucleotide molecules include at least one segment of 19 or more contiguous nucleotides cloned from or otherwise identified from the target gene in either anti- sense or sense orientation, whereby the polynucleotide molecules permeate the interior of the plant and induce modulation of the target gene. The conditioning and polynucleotide application can be performed separately or in a single step. When the conditioning and polynucleotide application are performed in separate steps, the conditioning can precede or can follow the polynucleotide application within minutes, hours, or days. In some embodiments more than one conditioning step or more than one polynucleotide molecule application can be performed on the same plant. In embodiments of the method, the segment can be cloned or identified from (a) coding (protein-encoding), (b) non-coding (promoter and other gene related molecules), or (c) both coding and non-coding parts of the target gene. Non-coding parts include DNA, such as promoter regions or the RNA transcribed by the DNA that provide RNA regulatory molecules, including but not limited to: introns, 5' or 3' untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, and riboswitches.
The following examples are included to demonstrate examples of certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1. Polynucleotides related to the herbicide target genes of johnsongrass (Sorghum halepense)
Polynucleotides were isolated from johnsongrass and sequenced and those identified as noncoding or coding regions of herbicide target genes acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS large subunit and ALS small subunit, also known as
acetohydroxyacid synthase, AHAS), dihydropteroate synthetase (DHPS), 5- enolpymvylshikimate-3 -phosphate synthase (EPSPS), glutamine synthetase (GS2), 4- hydroxyphenyl-pyruvate-dioxygenase (HPPD), phytoene desaturase (PDS),
protoporphyrinogen IX oxidase (PPOX) were selected. These are shown as SEQ ID NO: l- 120.
Polynucleotide molecules were extracted from johnsongrass tissues by methods standard in the field, for example, total RNA was extracted using Trizol Reagent (Invitrogen Corp, Carlsbad, CA Cat. No. 15596-018), following the manufacturer's protocol or modifications thereof by those skilled in the art of polynucleotide extraction that may enhance recover or purity of the extracted RNA. Briefly, starting with approximately 1 gram of ground plant tissue for extraction. Prealiquot 10 milliliters (mL) Trizol reagent to 15 mL conical tubes. Add ground powder to tubes and shake to homogenize. Incubate the homogenized samples for 5 minutes (min) at room temperature (RT) and then add 3 mL of chloroform. Shakes tubes vigorously by hand for 15-30 seconds (sec) and incubate at RT for 3 min. Centrifuge the tubes at 7,000 revolutions per minute (rpm) for 10 min at 4 degrees C (centigrade). Transfer the aqueous phase to a new 1.5 mL tube and add 1 volume of cold isopropanol. Incubate the samples for 20-30 min at RT and centrifuge at 10,000 rpm for 10 min at 4 degrees C. Wash pellet with Sigma-grade 80 percent ethanol. Remove the supernatant and briefly air-dry the pellet. Dissolve the RNA pellet in approximately 200 microliters of Diethylpyrocarbonate (DEPC) treated water. Heat briefly at 65C to dissolve pellet and vortex or pipet to resuspend RNA pellet. Adjust RNA concentraiton to 1-2 microgram/microliter. RNA was used to make cDNA libraries by standard methods that were then sequenced.
Genomic DNA (gDNA) was extracted using EZNA SP Plant DNA Mini kit (Omega Biotek, Norcross GA, Cat#D5511) and Lysing Matrix E tubes (Q-Biogen, Cat#6914), following the manufacturer's protocol or modifications thereof by those skilled in the art of polynucleotide extraction that may enhance recover or purity of the extracted DNA. Briefly, aliquot ground tissue to a Lysing Matrix E tube on dry ice, add 800μ1 Buffer SP1 to each sample, homogenize in a bead beater for 35-45sec, incubate on ice for 45-60 sec, centrifuge at > 14000 rpm for lmin at RT, add 10 microliter RNase A to the lysate, incubate at 65 °C for lOmin, centrifuge for lmin at RT, add 280μ1 Buffer SP2 and vortex to mix, incubate the samples on ice for 5min, centrifuge at >10,000g for 10 min at RT, transfer the supernatant to a homogenizer column in a 2ml collection tube, centrifuge at 10,000g for 2 min at RT, transfer the cleared lysate into a 1.5ml microfuge tube, add 1.5 volumes Buffer SP3 to the cleared lysate, vortex immediately to obtain a homogeneous mixture, transfer up to 650μ1 supernatant to the Hi-Bind column, centrifuge at 10,000g for lmin, repeat, apply ΙΟΟμΙ 65 °C Elution Buffer to the column, centrifuge at 10,000g for 5min at RT.
Next-generation DNA sequencers, such as the 454-FLX (Roche, Branford, CT), the
SOLiD (Applied Biosystems, ), and the Genome Analyzer (HiSeq2000, Illumina, San Diego, CA) are used to provide polynucleotide sequence from the DNA and RNA extracted from the plant tissues. Raw sequence data is assembled into contigs. The contig sequence is used to identify trigger molecules that can be applied to the plant to enable regulation of the gene expression. SEQ ID NO: 1-120 (summarized in Table 1) contains the target cDNA and gDNA sequence contigs from the various herbicide target genes of johnsongrass.
Table 1. Johnsongrass herbicide target gene sequences and fragments SEQ ID NO: 1-120.
ALS small cDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
ALS small gDNAContig
DHPS cDNAContig
DHPS cDNAContig
DHPS gDNAContig
DHPS gDNAContig
DHPS gDNAContig
DHPS gDNAContig
DHPS gDNAContig
EPSPS cDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
EPSPS gDNAContig
GS2 cDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
GS2 gDNAContig
HPPD cDNAContig
HPPD gDNAContig
HPPD gDNAContig
HPPD gDNAContig
HPPD gDNAContig
HPPD gDNAContig
HPPD gDNAContig
PDS cDNAContig
PDS gDNAContig
PDS gDNAContig 100 PDS gDNAContig
101 PDS gDNAContig
102 PDS gDNAContig
103 PDS gDNAContig
104 PDS gDNAContig
105 PDS gDNAContig
106 PPOX cDNAContig
107 PPOX gDNAContig
108 PPOX gDNAContig
109 PPOX gDNAContig
110 PPOX gDNAContig
111 PPOX gDNAContig
112 PPOX cDNAContig
113 PPOX cDNAContig
114 PPOX gDNAContig
115 PPOX gDNAContig
116 PPOX gDNAContig
117 PPOX gDNAContig
118 PPOX gDNAContig
119 PPOX gDNAContig
120 PPOX gDNAContig
Example 2. Polynucleotides of the invention related to trigger molecules of the johnsongrass herbicide target genes.
The gene sequences and fragments of SEQ ID NO: 1-120 were selected into short polynucleotide lengths of 30 contiguous nucleotides as shown in Table 2, SEQ ID NO: 121- 386. These polynucleotides are tested to select an efficacious trigger to any of the herbicide target gene sequence regions. The trigger polynucleotides are constructed as sense or anti- sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids and combined with an organosilicone based transfer agent and nonpolynucleotide herbicide to provide a new herbicidal composition. The polynucleotides are combined into sets of two to three polynucleotides per set, using 4-8 nM of each polynucleotide. Each polynucleotide set is prepared with the organosilicone transfer agent and applied to a johnsongrass plant or to a field of crop plants containing johnsongrass plants in combination with a nonpolynucleotide herbicide that targets one or more of the enzymes of the herbicide target genes, or followed by the nonpolynucleotide herbicide treatment one to three days later. The effect is measured as stunting the growth and/or killing of the plant and is measured 8-14 days after treatment with the herbicidal composition. The most efficacious trigger sets are identified and the individual polynucleotides are tested in the same methods as the sets are and the most efficacious single polynucleotide is identified. By this method it is possible to identify one oligonucleotide or several oligonucleotides that effect plant sensitivity to nonpolynucleotide herbicide.
It is contemplated that additional 19-30 polynucleotides can be selected from the sequences of SEQ ID NO: 1-120 that are specific for a herbicide target gene in johnsongrass or include activity against a few related weed species, for example, Sorghum bicolor and Sorghum Sudanese.
Table 2. Polynucleotides SEQ ID NO: 121-386.
156 GGGCCTTGGCAACTTCCCCGGCGACGACCC ALS
157 GGGTGATGTGTTATTTATGTGATGTTCTCC ALS
158 GGGTGATGTGTTATTTATGTGATGTTCTCC ALS
159 GGGCCTTGGCAACTTCCCCGGTGACGACCC ALS
160 GGAGCGGAGACGGAGGAAGGCGTGGCACCC ALS
161 GGCCGCCAAATAGGCCACCACGGCACCACC ALS
162 GGCCAAGAGCATTTGCACGTCACCAATGCC ALS
163 GGATAATAGCCGATACAGTAGCTGTCAGCC ALS
164 GGGCGGTGAACACTGCGACTGACCCGCCCC ALS
165 GGACTGGATGCGCCTGAAAATCACCGCACC ALS
166 GGCGTCCCCATCGAAACCATCGCACAAGCC ALS
167 GGGTGATGGACCCTTGGGGCATTGTTAACC ALS
168 GGAGGCATCCAAAGATTTGGGCCGCAGCCC ALS
169 GG AC AAGC GGGC AG AGTTC GGGTT ACTGC C ALS
170 GGCCGACCGTTTCCGCCCACTCGGCAGGCC ALS
171 GGCCGCGCAGGACGTCGCGGCATCGACACC ALS
172 GGTTGCCGACTCACTGTCGAGCCTTGATCC ALS
173 GGACCGCGAAGAAGACTGATTCGTGCGCCC ALS
174 GGTGTCAACACCGCAGCGATTGCTCATCCC ALS
175 GGTTTTGGGGTGACGACCCATGGTGGCACC ALS
176 GGGCATCAACGCCATTCGGTCGGCCATGCC ALS
177 GGACAGCACGACCTGCCCGATATCTCTGCC ALS
178 GGGTGCGTCAGTTACAGCGGATGAAAAACC ALS
179 GGTGAAGAAACGCCCGAGGAAGAAATCTCC ALS
180 GGAGCCAGTAATGATGAAATCACGATCGCC ALS
181 GGCGACGAGGACAGCAACGATGATGAGGCC ALS
182 GGGTGACGTTCCCTAGATCCCAAGACAACC ALS
183 GGCATTGAAACGAGCTTCCGTGAGGAGACC ALS
184 GGTATCTCCAAGCTGCGTTGGTCAATTTCC ALS
185 GGTGCCATTGATGTCGTGGGGAGGAAAACC ALS
186 GGGGGGCATATCGTCGATGACGACGAGGCC ALS
187 GGCCGCGCTCCAAATAAAACTGACGGCACC ALS
188 GGCCAGAGTGGAGTTGTCAAGAACCTCTCC ALS
189 GGTCAACTACCACGGACTTGATATCAATCC ALS
190 GGCCGTTCATGATCGCCTACCAGGATTTCC ALS
191 GGTGGCAATTACGCAGCTTCGCTGCGTTCC ALS
192 GGAGATACCGACGTGAAGGTCTCTGAGCCC ALS
193 GGACATGGCATCAATCCCGGTGATGACGCC ALS
194 GGGCTTACCCTTCTCCAGTGCGTGGATGCC ALS
195 GGGTTCACCGTTGCCAATGACGTCACTGCC ALS
196 GGGTGCTGACACCTTCTGCCCGCTGGGGCC ALS
197 GGGTATCACACCGTCGGGGGGCTCATAGCC ALS
198 GGACTGTCGGTGAACCTGCCGGAAAAAGCC ALS
199 GGCATGACCGCGATGAGTTGGGTGGACGCC ALS
200 GGGGGACAATCGAAGAAGGTTCTCAAGACC ALS
201 GGTC CTG AC AC CG AT AT AGC CGC AATG AC C ALS
202 GGAGCGGAGACGGAGGAAGGCGTGGCACCC ALS
203 GGCCGCCAAATAGGCCACCACGGCACCACC ALS 204 GGCCAAGAGCATTTGCACGTCACCAATGCC ALS
205 GGATAATAGCCGATACAGTAGCTGTCAGCC ALS
206 GGTCGGTGTCAGTGCCGTTTACTCTGGGCC ALS
207 GGAGGGAGGCCTCCACGCACATCCCCCTCC ALS
208 GGGCGCACCTCCTGGCCGCACGGCGCGCCC ALS
209 GGGCCTTGGCAACTTCCCCGGCGACGACCC ALS
210 GGGTGATGTGTTATTTATGTGATGTTCTCC ALS
211 GGTTACAGCATGCTAGTTGTTTAGACTTCC ALS small
212 GGCCCCCGCTGCCGTGTCGGCGGTCGCCCC ALS small
213 GGTGACCAAACAGCTCAATAAGATTATTCC ALS small
214 GGAGCGGAGACGGAGGAAGGCGTGGCACCC ALS small
215 GGCCGCCAAATAGGCCACCACGGCACCACC ALS small
216 GGCCAAGAGCATTTGCACGTCACCAATGCC ALS small
217 GGATAATAGCCGATACAGTAGCTGTCAGCC ALS small
218 GGAACAGAAGGTATTAAAAGGGTATTACCC DHPS
219 GGTCTGATAGAAGTCTCATCATGGGGATCC DHPS
220 GGAGGAAAGTTTCAACCAGTGGAAGCTGCC DHPS
221 GGTGAGAGAAGCAGAGTTATCTGGGATTCC DHPS
222 GGAGGAAAGTTTCAACCAGTGGAAGCTGCC DHPS
223 GGTGAGAGAAGCAGAGTTATCTGGGATTCC DHPS
224 GGTCATTTGTTTTAGCACCTCTTGTTGACC DHPS
225 GGAGGTAAGTTTCAACAAGTGGAAGCTGCC DHPS
226 GGTGAGAGAAGCAGAGTTATCTGGGATTCC DHPS
227 GGCACCTCCTAGTCTTTGCTGTCTTCATCC DHPS
228 GGGTGCTAGCTTAAAAAAAAGATTAACACC DHPS
229 GGC ATTT ACGC C AGT AATTGT AC AAGG AC C DHPS
230 GGCGCCGGCGCCTCAGCTTGTACGGCCTCC DHPS
231 GGCACTCAGGGTCTTCCTGATCTTGTTCCC DHPS
232 GGAGTGCTGATGGGAATATCCCTTGTAGCC DHPS
233 GGAACAGAAGGTATTAAAAGGGTATTACCC DHPS
234 GGTCTGAGAGAACTCTCATCATGGGGATCC DHPS
235 GGAGGAAAGTTTCAACCAATGGAAGCTGCC DHPS
236 GGAAAAGTTTTTTGGGTGAAATATGCAACC DHPS
237 GGGCTCTCTGTCGAAGCAGACAAAGTTGCC EPSPS
238 GGCTCCATCAGCAGTCAGTACTTGAGTGCC EPSPS
239 GGTCAAAAATACAAGTCCCCCAAAAATGCC EPSPS
240 GGCTATTGATGTTAACATGAACAAAATGCC EPSPS
241 GGCCCAACAGCTATCAGAGACGTGGCGTCC EPSPS
242 GGCTATTGATGTTAACATGAACAAAATGCC EPSPS
243 GGGGCATTTAATGCAGCAAAATGACAGGCC EPSPS
244 GGAAAAGGTACTTGATTGGTTTTTTGTGCC EPSPS
245 GGACCGAGACTAGCGTTACTGTTACTGGCC EPSPS
246 GGACAAGGCACTTGATTGGTTTTTTGCCCC EPSPS
247 GGCAGGCGCCGAGGAGATCGTGCTGCAGCC EPSPS
248 GGTGGGTGTCGCCCTATGCCCCTATCGGCC EPSPS
249 GGGCTCTCTGTCGAAGCAGACAAAGTTGCC EPSPS
250 GGTCATCCCTAACTAGCAAACCATGTTTCC EPSPS
251 GGCGGGCGCCGAGGAGATCGTGCTGCAGCC EPSPS 252 GGTGGGTGTCGCCCTATGCCCCTATCGGCC EPSPS
253 GGGCTCTCTGTCGAAGCAGACAAAGTTGCC EPSPS
254 GGTCATCCCTAACTAGCAAACCATGTTTCC EPSPS
255 GGCTCCATCAGCAGTCAGTACTTGAGTGCC EPSPS
256 GGCTATTGATGTTAACATGAACAAAATGCC EPSPS
257 GGGGCATTGAATGCAGCAAAATGACAGGCC EPSPS
258 GGCGGGCGCCGAGGAGATCGTGCTGCAGCC EPSPS
259 GGTGGGTGTCGCCCTATGCCCCTATCGGCC EPSPS
260 GGGCTCTCTGTCGAAGCAGACAAAGTTGCC EPSPS
261 GGCTCCATCAGCAGTCAGTACTTGAGTGCC EPSPS
262 GGCTATTGATGTTAACATGAACAAAATGCC EPSPS
263 GGGGCATTGAATGCAGCAAAATGACAGGCC EPSPS
264 GGTGAACTAGACTGATGACTGGGCGGGTCC EPSPS
265 GGATCCATCAGGCCCGCCTCGAACCCGGCC EPSPS
266 GGCGGGCGCCGAGGAGATCGTGCTGCAGCC EPSPS
267 GGGCTCTCTGTCGAAGCAGACAAAGTTGCC EPSPS
268 GGTCATCCCTAACTAGCAAACCATGTTTCC EPSPS
269 GGCTCCATCAGCAGTCAGTACTTGAGTGCC EPSPS
270 GGAAAAGGTACTTGATTGGTTTTTTGTGCC EPSPS
271 GGACCGAGACTAGCGTTACTGTTACTGGCC EPSPS
272 GGCGCCTCGCCGGGGTTCAAGGTCATGGCC GS2
273 GGG AG AAG AC AGTG AAGTC ATTCT AT AC C C GS2
274 GGCACAGGGCTGCGCAAATTTTTAGTGACC GS2
275 GGAACTCTATAAATATAAATCAAATCAACC GS2
276 GGATTTGGAGAGGGGTTTTGGGAGACCGCC GS2
277 GGCCCGGTGACCGATCCCAGCAAGCTGCCC GS2
278 GGTAGGTACGGTATTGAGCAGGAGTACACC GS2
279 GGCAACTTCTTTTGTAACCCTCAAGCTACC GS2
280 GGCTGCTCTGTTCGTGTGGGGCGAGATACC GS2
281 GGAAAAAAGTTCAATTTATCTCTCCCAACC GS2
282 GGTGATCTAACATGTAAAATGTAAGACTCC GS2
283 GGTGC CGGC GC AC AC AC C AACT AC AGC AC C GS2
284 GGCAGGCACGAGACCGCCGACATCAACACC GS2
285 GGATTGATGTGAATCCGACTAAACAAGGCC GS2
286 GGGGCCAACAAATTAAATCTGAGATATCCC GS2
287 GGTGC CGGC GC AC AC AC C AACT AC AGC AC C GS2
288 GGCAGGCACGAGACCGCCGACATCAACACC GS2
289 GGCCCGGTGACCGATCCCAGCAAGCTGCCC GS2
290 GGTAGGTACGGTATTGAGCAGGAGTACACC GS2
291 GGCAACTTCTTTTGTAACCCTCAAGCTACC GS2
292 GGCCCGGTGACCGATCCCAGCAAGCTGCCC GS2
293 GGCCCTATAATGTGCTTGGTTTCCCGTTCC GS2
294 GGAAAAAAGTTTAATTTATCTCTCCCAGCC GS2
295 GGACAGGGTAATTAACCCAACAATGCCTCC GS2
296 GGACAGTGTCCTATGGTTTGGTGGGGTGCC GS2
297 GGTGCTTGCTCTGATGCTGGTAATTGTACC GS2
298 GGTCTGGGTGGATGCATCATGCATCATGCC GS2
299 GGCTCATGTTGTGGGTGGATGCGTCATGCC GS2 300 GGTAGGTACGGTATTGAGCAGGAGTACACC GS2
301 GGCAACTTCTTTTGTAACCCTCAAGCTACC GS2
302 GGTGC CGGC GC AC AC AC C AACT AC AGC AC C GS2
303 GGCAGGCACGAGACCGCCGACATCAACACC GS2
304 GGCTGGGCACCCATATTTCTCCCTCGGACC GS2
305 GGCTCCTCCAGAAATGGCTTACGGTGGGCC GS2
306 GGAGCCACCAAGCACTGCACGGCCCCCTCC GS2
307 GGGCCGATTGGCCGGATCGAATACTTCTCC GS2
308 GGTCAGTCAAGCCCTGACAGCGGCCGGCCC GS2
309 GGCAAGCATTCCACGCAGTGTCTCTCAGCC GS2
310 GGTTCCACTAGTCTTCTTGGTCTAGTGACC GS2
311 GGGGCCAACAAATTAAACTCTGAGATATCC GS2
312 GGGTTTGCATGCCTCTGAAGGATCAGGCCC GS2
313 GGCAACGTGCCGGAGCTGGCGCCGGCGGCC HPPD
314 GGAGAACGTGCTGCTCCCACTCAACGAGCC HPPD
315 GGCCCCGGCGTGCAGCACATGGCGCTGGCC HPPD
316 GGCAACGTGCCGGAGCTGGCGCCGGCGGCC HPPD
317 GGAGAACGTGCTGCTCCCACTCAACGAGCC HPPD
318 GGCCCCGGCGTGCAGCACATGGCGCTGGCC HPPD
319 GGTCTCCAGGGAACAAGAAGTTGCTGCGCC HPPD
320 GGCCAGGTCGCCGCCAATTGCCGTCCAGCC HPPD
321 GGTCTCCAGGGAACAAGAAGTTGCTGCGCC HPPD
322 GGGCGCCCTCGCTTTCCTCTTCACGGCGCC HPPD
323 GGCGCCGACGCCGCCACGGCCTCGCTGCCC HPPD
324 GGAGAACGTGCTGCTCCCACTCAACGAGCC HPPD
325 GGCCCCGGCGTGCAGCACATGGCGCTGGCC HPPD
326 GGGCGCCCTCGCTTTCCTCTTCACGGCGCC HPPD
327 GGGCTTCATATCTTTTTTGGAGCTTATCCC PDS
328 GGTTTACAAAACTGTCCCAAACTGTGAACC PDS
329 GGTGGTTC C AATC AATC GGTT AAATC ATC C PDS
330 GGGGCGTCTAGCGCCTTGCACGGGTGACCC PDS
331 GGTTGCGCTATCGTTCATGTTTGAATGTCC PDS
332 GGTGGTTCCTATCAATCGGTTAATTCATCC PDS
333 GGAAGATTTGTCCATTCTGCTTGGTGCCCC PDS
334 GGACCAAGAAAGCATCAGAACAATAATACC PDS
335 GGATATACTCCTAGTAGTCTGTAGTGCGCC PDS
336 GGGCTCCCCCGCCTCCACGACACTGCCTCC PDS
337 GGTGCTACGAAATTGTCTAGAACGAGGTCC PDS
338 GGCTTCATGAACTGTGGGTCTAATGGCTCC PDS
339 GGACGGAGGCCCATGTGAGCAAGTTGGGCC PDS
340 GGCAGTGGTACTAGTATCCGAAATGTGACC PDS
341 GGTT AACT ATATATTTTTGTAGATGTCGCC PDS
342 GGCAACTCCCGACGGATCTATTGCCTCCCC PDS
343 GGGGAAGCTTATCCCCCCTCTTATCGAGCC PDS
344 GGTCTCTGCTCCGGTAGCGGCGCGTCTCCC PDS
345 GGTTGCGCTATCGTTCATGTTTGAATGTCC PDS
346 GGTGGTTCCTATCAATCGGTTAATTCATCC PDS
347 GGAAGATTTGTCCATTCTGCTTGGTGGCCC PDS 348 GGACCAAGAAAGCATCAGAACAATAATACC PDS
349 GGCCTACCTTTATGGATCGAATAATCAACC PDS
350 GGCCGGCCGGCCGGCCGACGGACCGAGACC PDS
351 GGCGGCGACCAGGGTGATCGGATCCAAGCC PDS
352 GGCACGCTGAAGAAGCTTGTGAACGAGTCC PDS
353 GGCGGCAACATCACCACCGTCGAGCGCCCC PPOX
354 GGGCCGGTCTCGGCGCGCTTGGCATCCGCC PPOX
355 GGAGGTGCTACAAACACAGGAATTGTTTCC PPOX
356 GGAGGTGCTACAAACACAGGAATTGTTTCC PPOX
357 GGTTAGCAATACTCTGCCAAAGCTATTGCC PPOX
358 GGGGACGTGCTTGTCACGGAGGCCCGCGCC PPOX
359 GGGCCGGTCTCGGCGCGCTTGGCATCCGCC PPOX
360 GGAGGTGCTACAAACACAGGAATTGTTTCC PPOX
361 GGTTAGCAATACTCTGCCAAAGCTATTGCC PPOX
362 GGGCCGGTCTCGGCGCGCTTGGCATCCGCC PPOX
363 GGTAGAAGCATCAAATGAAAAGAATTGCCC PPOX
364 GGATAGTTCTGTTGGAAAAGTTGAAGTCCC PPOX
365 GGGTTTCTCTGGGATGAAGGAGCGAACACC PPOX
366 GGACATTACTTCACAATGAGTATCACTTCC PPOX
367 GGATCACGGTTCGCAGGTCAGCTTGTGGCC PPOX
368 GGAGACCAGCCTGAACTTGCTTCCGAAACC PPOX
369 GGTATATGGCATTCCAGAATTCCGTCTTCC PPOX
370 GGTCCACCGCCGTGTCACGGACACGGCTCC PPOX
371 GGTTATTGAGGAAAATTTGGATCAGCTGCC PPOX
372 GGTGGCATTAACCCTGCATCATGATTTTCC PPOX
373 GGTAGAAGCATCAAATGAAAAGAATTGCCC PPOX
374 GGATAGTTCTGTTGGAAAAGTTGACGTCCC PPOX
375 GGTCCTAGCTCAGTTGGTTGAGGGTATGCC PPOX
376 GGCTCTCGCCGCCGCCGCCGCCTCGAGGCC PPOX
377 GGTCCACCGCCGTGTCACGGACACGGCTCC PPOX
378 GGTTATTGAGGAAAATTTGGATCAGCTGCC PPOX
379 GGTGGCATTAACCCTGCATCATGATTTTCC PPOX
380 GGTAGAAGCATCAAATGAAAAGAATTGCCC PPOX
381 GGATAGTTCTGTTGGAAAAGTTGAAGTCCC PPOX
382 GGTCCTAGCTCAGTTGGTTGAGGGTATGCC PPOX
383 GGACATTACTTCACAATGAGTATCACTTCC PPOX
384 GGACTCACGGCTGCTGAAGAGCTCGCCTCC PPOX
385 GGCGGCCGCTTAGAAAACGCTGAGTTATCC PPOX
386 GGGCGGCGGCTAATGCCACCTGGTTGAACC PPOX
Example 3. Methods used in the invention related to treating plants or plant parts with a topical mixture of the trigger molecules.
Johnsongrass plants are grown in the greenhouse (30 / 20 C day/night T; 14 hour photoperiod) in 4 inch square pots containing Sun Gro® Redi-Earth and 3.5 kg/cubic meter Osmocote® 14-14-14 fertilizer. When the plants at 5 to 10 cm in height are pre-treated with a mixture of single-strand antisense or double-strand polynucleotides (ssDNA ro dsRNA targeting one or more of the herbicide target gene sequences from SEQ ID NO: 1-120) at 16 nM, formulated in 10 millimolar sodium phosphate buffer (pH 6.8) containing 2%
ammonium sulfate and 0.5% Silwet L-77. Plants are treated manually by pipetting 10 μΐ^ οΐ polynucleotide solution on fully expanded mature leaves, for a total of 40 microliters of solution per plant. Twenty-four and forty-eight hours later, the plants are treated with and effective dose of the nonpolynucleotide herbicide corresponding to the herbicide target gene in which the polynucleotides have homology. Four replications of each treatment is conducted. Plant height is determined just before treatment and at intervals upto twelve days after herbicide treatments to determine effect of the polynucleotide and herbicide treatments.
Example 4. A method to control johnsongrass in a field.
A method to control johnsongrass in a field comprises the use of trigger
polynucleotides that can modulate the expression of one or more herbicide target genes in johnsongrass. In Table 2, an analysis of herbicide target gene sequences provided a collection of 30-mer polynucleotides that can be used in compositions to affect the growth or develop or sensitivity to a polynucleotide herbicide to control multiple weed species in a field. A composition containing 1 or 2 or 3 or 4 or more of the polynucleotides of Table 2 or fragments thereof would enable broad activity of the composition against the herbicide resistant johnsongrass species or multiple Sorghum weed species that occur in a field environment.
The method includes creating a composition that comprises components that include at least one polynucleotide of Table 2 or fragment thereof or any other effective gene expression modulating polynucleotide essentially identical or essentially complementary to SEQ ID NO: 1-120, a transfer agent that mobilizes the polynucleotide into a plant cell and nonpolynucleotide herbicide. The polynucleotide of the composition includes a dsRNA, ssDNA or dsDNA or a combination thereof. A composition containing a polynucleotide can have a use rate of about 1 to 30 grams or more per acre depending on the size of the polynucleotide and the number of polynucleotides in the composition. The composition may include one or more additional herbicides as needed to provide effective multi-species weed control in addition to control of johnsongrass and related weed species. A field of crop plants in need of weed plant control is treated by spray application of the composition. The composition can be provided as a tank mix, a sequential treatment of components (generally the polynucleotide followed by the nonpolynucleotide herbicide), a simultaneous treatment or mixing of one or more of the components of the composition from separate containers or as a premix of all of the components of the herbicidal composition. Members of the
nonpolynucleotide herbicide families include but are not limited to amide herbicides, aromatic acid herbicides, arsenical herbicides, benzothiazole herbicides,
benzoylcyclohexanedione herbicides, benzofuranyl alkylsulfonate herbicides, carbamate herbicides, cyclohexene oxime herbicides, cyclopropylisoxazole herbicides, dicarboximide herbicides, dinitroaniline herbicides, dinitrophenol herbicides, diphenyl ether herbicides, dithiocarbamate herbicides, halogenated aliphatic herbicides, imidazolinone herbicides, inorganic herbicides, nitrile herbicides, organophosphorous herbicides, oxadiazolone herbicides, oxazole herbicides, phenoxy herbicides, phenylenediamine herbicides, pyrazole herbicides, pyridazine herbicides, pyridazinone herbicides, pyridine herbicides,
pyrimidinediamine herbicides, pyrimidinyloxybenzylamine herbicides, quaternary ammonium herbicides, thiocarbamate herbicides, thiocarbonate herbicides, thiourea herbicides, triazine herbicides, triazinone herbicides, triazole herbicides, triazolone herbicides, triazolopyrimidine herbicides, uracil herbicides, and urea herbicides. Treatment of the plants in the field can occur as often as needed to provide weed control and the components of the composition can be adjusted to target specific weed species or weed families.

Claims

We Claim:
1. A method of Sorghum weed species plant control comprising: treating a Sorghum weed species plant or a part of said plant in need of control with a first herbicidal composition comprising a polynucleotide, an organosilicone surfactant concentration of about 0.2 percent or greater, and an effective dose of a nonpolynucleotide herbicide, wherein said
polynucleotide is at least 19 contiguous polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum weed species gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120, wherein said treated plant is more sensitive to said nonpolynucleotide herbicide relative to a similar plant treated with a second herbicidal composition not containing said polynucleotide.
2. The method of claim 1, wherein said Sorghum weed species is selected from the group consisting of Sorghum halepense, Sorghum bicolor and Sorghum Sudanese.
3. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-25 and said nonpolynucleotide herbicide is selected from the group consisting of aryloxyphenoxypropionates, cyclohexanediones and phenylpyrazoline.
4. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 26-44 and said nonpolynucleotide herbicide is selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
5. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 45-59 and said nonpolynucleotide herbicide is selected from the group consisting of sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl(thio)benzoates, and sulfonylaminocarbonyl-triazolinones.
6. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 60-66 and said nonpolynucleotide herbicide is selected from the group consisting of sulfonamides and asulam.
7. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 67-74 and said nonpolynucleotide herbicide is glyphosate.
8. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 75-89 and said nonpolynucleotide herbicide is glufosinate.
9. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 90-96 and said nonpolynucleotide herbicide is selected from the group consisting of triketones, isoxazoles, and pyrazoles.
10. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 97-105 and said nonpolynucleotide herbicide is selected from the group consisting of pyridazinones, pyridinecarboxamides, beflubutamid, fluridone, flurochloridone and flurtamone.
11. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides in length and essentially identical or essentially complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 106-120 and said nonpolynucleotide herbicide is selected from the group consisting of acifluorfen-Na, bifenox, chlomethoxyfen, fluoroglycofen-ethyl, fomesafen, halosafen, lactofen, oxyfluorfen, fluazolate, pyraflufen-ethyl, cinidon-ethyl, flumioxazin, flumiclorac- pentyl, fluthiacet-methyl, thidiazimin, oxadiazon, oxadiargyl, azafenidin, carfentrazone-ethyl, sulfentrazone, pentoxazone, benzfendizone, butafenacil, pyrazogyl, and profluazol.
12. The method of claim 1, wherein said polynucleotide is selected from the group consisting of sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA, or dsDNA/RNA hybrids.
13. The method of claim 1, wherein said polynucleotide is at least 19 contiguous nucleotides, 20 contiguous nucleotides or 21 contiguous nucleotides in length and at least 85 percent identical or complementary to a segment of said Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120.
14. The method of claim 1, wherein said polynucleotide is at least 19 contiguous
polynucleotides selected from the group consisting of SEQ ID NO: 121-386.
15. The method of claim 1, wherein said polynucleotide is at least 85 percent homologous or complementary to polynucleotides selected from the group consisting of SEQ ID NO: 121- 386.
16. The method of claim 1, wherein said herbicidal composition comprises any combination of two or more of said polynucleotides, wherein said polynucleotide is at least 19
polynucleotides in length and at least 85 percent identical or complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120, and said herbicidal composition comprises any combination of two or more nonpolynucleotide herbicides that inhibit the activity of two or more proteins selected from the group consisting of ACCase, ALS large subunit, ALS small subunit, DHPS, EPSPS, GS2, HPPD, PDS, PPOX, and wherein said treated Sorghum halepense plant is more sensitive to the two or more nonpolynucleotide herbicides relative to a similar plant treated with a herbicidal composition not containing said polynucleotides.
17. The method of claim 1, wherein said herbicidal composition further comprises one or more nonpolynucleotide herbicide selected from the group consisting of: 5-Diarylpyrazole herbicides, 2-Thiopyrimidine herbicides, 3-CF3-Benzene herbicides, Acetamide herbicides, Amide herbicides, Aminoacrylate herbicides, Aminotriazine herbicides, Aromatic acid herbicides, Arsenical herbicides, Arylaminopropionic acid herbicides, Arylcarboxamide herbicides, Arylcyclodione herbicides, Aryloxyphenoxy-propionate herbicides,
Azolecarboxamide herbicides, Azoloazinone herbicides, Azolotriazine herbicides,
Benzamide herbicides, Benzenesulfonamide herbicides, Benzhydryl herbicides,
Benzimidazole herbicides, Benzofuran herbicides, Benzofuranyl Alkylsulfonate herbicides, Benzohydrazide herbicides, Benzoic acid herbicides, Benzophenylmethanone herbicides, Benzothiadiazinone herbicides, Benzothiazole herbicides, Benzothiazoleacetate herbicides, Benzoxazole herbicides, Benzoylcyclohexanedione herbicides, Benzyloxymethylisoxazole herbicides, Benzylpyrazole herbicides, Benzylpyridine herbicides, Benzylpyrimidone herbicides, Bipyridylium herbicides, Carbamate herbicides, Chloroacetamide herbicides, Chloroacetamide herbicides, Chlorocarbonic acid herbicides, Cyclohexanedione herbicides, Cyclohexene oxime herbicides, Cyclopropylisoxazole herbicides, Diarylether herbicides, Dicarboximide herbicides, Dihydropyrancarboxamide herbicides, Diketo-epoxide herbicides, Diketopiperazine herbicides, Dinitroaniline herbicides, Dinitrophenol herbicides,
Diphenylether herbicides, Diphenylfuranone herbicides, Dithiocarbamate herbicides, Fluoroalkene herbicides, Glyphosate herbicides, Halogenated aliphatic herbicides,
Hydantocidin herbicides, Hydroxypyrazole herbicides, Imidazolinone herbicides, Indazole herbicides, Indenedione herbicides, Inorganic herbicides, Isoxazole herbicides,
Isoxazolesulfone herbicides, Isoxazolidinone herbicides, Nicotinohydrazide herbicides, Nitrile herbicides, Nitrile-amide herbicides, Nitropyrazole herbicides, N-phenylphthalimide herbicides, Organoarsenical herbicides, Organophosphates herbicides, Organophosphorus herbicides, Oxabicycloheptane herbicides, Oxadiazole herbicides, Oxadiazolebenzamide herbicides, Oxadiazolone herbicides, Oxazole herbicides, Oxazolidinedione herbicides, Oxyacetamide herbicides, Phenoxy herbicides, Phenoxyalkyne herbicides,
Phenoxycarboxylic acid herbicides, Phenoxypyridazinol herbicides, Phenylalkanoate herbicides, Phenylcarbamate herbicides, Phenylenediamine herbicides, Phenylethylurea herbicides, Phenylimidazole herbicides, Phenylisoxazole herbicides, Phenylpyrazole herbicides, Phenylpyrazoline herbicides, Phenylpyridazine herbicides, Phenylpyridine herbicides, Phenylpyrrolidone herbicides, Phosphinic acid herbicides, Phosphonate herbicides, Phosphoroamidate herbicides, Phosphorodithioate herbicides, Phthalamate herbicides, Propionamide herbicides, Pyrazole herbicides, Pyrazole-arylether herbicides, Pyrazolium herbicides, Pyridazine herbicides, Pyridazinone herbicides, Pyridine herbicides, Pyridinecarboxamide herbicides, Pyridinecarboxylic acid herbicides, Pyridinone herbicides, Pyridyl-benzylamide herbicides, Pyridyl-ether-carboxamide herbicides, Pyrimidinecarboxylic acid herbicides, Pyrimidinediamine herbicides, Pyrimidinedione herbicides, Pyrimidinetrione herbicides, Pyrimidinone herbicides, Pyrimidinyl(thio)benzoate herbicides,
Pyrimidinyloxybenzylamine herbicides, Pyrimidylmethanol herbicides, Pyrrolidone herbicides, Quaternary Ammonium herbicides, Quinoline-carboxylic acid herbicides, Quinoxaline herbicides, Semicarbazone herbicides, Sulfonamide herbicides, Sulfonylamino- carbonyl-triazolinone herbicides, Sulfonylurea herbicides, Sulfonylurea herbicides,
Tetrazolinone herbicides, Thiadiazole herbicides, Thiatriazine herbicides, Thienopyrimidine herbicides, Thiocarbamate herbicides, Thiocarbonate herbicides, Thiourea herbicides, Tolyltriazole herbicides, Triazine herbicides, Triazinedione herbicides, Triazine-sulfonanilide herbicides, Triazinone herbicides, Triazole herbicides, Triazolecarboxamide herbicides, Triazoleimine herbicides, Triazolinone herbicides, Triazolone herbicides, Triazolopyrimidine herbicides, Triketone herbicides, Uracil herbicides, and Urea herbicides.
18. The method of claim 1, wherein said organosilicone surfactant concentration is about 0.2 percent to about 2.0 percent in said herbicide composition.
19. A herbicidal composition comprising an admixture of a polynucleotide, an
organosilicone surfactant concentration of about 0.2 percent or greater, and an effective dose of a nonpolynucleotide herbicide, wherein said polynucleotide is at least 19 polynucleotides in length and at least 85 percent identical or complementary to a segment of a Sorghum halepense gene polynucleotide selected from the group consisting of SEQ ID NO: 1-120, wherein a plant treated with said herbicidal composition is more sensitive to the herbicide contained in the herbicidal composition relative to a similar plant treated with a herbicidal composition not containing the polynucleotide.
20. The herbicidal composition of claim 19, wherein said polynucleotide is at least 19 contiguous polynucleotides selected from the group consisting of SEQ ID NO: 121-386.
21. The method of claim 19, wherein said polynucleotide is at least 85 percent homologous or complementary to polynucleotides selected from the group consisting of SEQ ID NO: 121- 386.
22. The herbicidal composition of claim 19, further comprising a pesticide, wherein said pesticide is selected from the group consisting of insecticides, fungicides, nematocides, bactericides, acaricides, growth regulators, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, and biopesticides.
23. The herbicidal composition of claim 19, comprising a premix or a tankmix combination.
EP14780250.8A 2013-03-13 2014-03-11 Methods and compositions for weed control Ceased EP2971185A4 (en)

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